The Molecular Probes® Handbook

A GUIDE TO FLUORESCENT PROBES AND LABELING TECHNOLOGIES

11th Edition (2010)

Molecular Probes™ Handbook

A Guide to Fluorescent Probes and Labeling Technologies
11th Edition (2010)

CHAPTER 1
Fluorophores
CHAPTER 17 and
Their for
Probes Amine-Reactive
Derivatives
Signal Transduction

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Identify compatible sets of fluorescent dyes and cell structure probes
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 SEVENTEEN
CHAPTER 17
Probes for Signal Transduction
17.1 Introduction to Signal Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 775

17.2 Calcium Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776

Inositol Triphosphate Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776
D-myo-1,4,5-Inositol Triphosphate and Caged D-myo-1,4,5-Inositol Triphosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776
Fluorescent Heparin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776
Caged Ca2+ and Caged Ca2+ Chelators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776
NP-EGTA: A Caged Ca2+ Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776
DMNP-EDTA: A Caged Ca2+ Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777
Diazo-2: A Photoactivatable Ca2+ Knockdown Reagent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777
Other Probes for Calcium Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777
Thapsigargin and Fluorescent Thapsigargin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777
Luminescent Calcium Analog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777
Data Table 17.2 Calcium Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778
Product List 17.2 Calcium Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778

17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins . . . . . . . 779
Protein Kinase Probes and Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779
Antibody Beacon™ Tyrosine Kinase Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779
Fluorescent Polymyxin B Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 780
Hypericin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 780
Protein Phosphatase Assay Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781
RediPlate™ 96 EnzChek® Tyrosine Phosphatase Assay Kits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781
RediPlate™ 96 EnzChek® Serine/Threonine Phosphatase Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781
Pro-Q® Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782
Adenylate Cyclase Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
Adenylate Cyclase Probe: BODIPY® FL Forskolin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
cAMP Chemiluminescent Immunoassay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
Nucleotide Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
Alexa Fluor® cAMP and Alexa Fluor® ATP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784
BODIPY® Ribonucleotide Di- and Triphosphates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784
Nonhydrolyzable BODIPY® ATP and GTP Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785
N-Methylanthraniloyl (MANT) Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785
Ethenoadenosine Nucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
Trinitrophenyl (TNP) Nucleotides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
Caged Nucleotides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
BzBzATP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
Data Table 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
Product List 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789

TheMolecular
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Chapter 17 — Probes for Signal Transduction

17.4 Probes for Lipid Metabolism and Signaling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 790

Phospholipase A1 and A2 Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791
PED-A1 Phospholipase A1 Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791
EnzChek® Phospholipase A1 Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792
Red/Green BODIPY® PC-A2 Ratiometric Phospholipase A2 Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792
EnzChek® Phospholipase A2 Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792
PED6 Phospholipase A2 Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793
Other BODIPY® Dye Phospholipase A Substrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793
Bis-Pyrenyl Phospholipase A Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
Singly Labeled Pyrenyl and NBD Phospholipase A2 Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
ADIFAB Indicator: A Different View of Phospholipase A Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
Phospholipase C Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795
EnzChek® Direct Phospholipase C Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795
Amplex® Red Phosphatidylcholine-Specific Phospholipase C Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795
Bacillus cereus PI-PLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796
Phospholipase D Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796
Amplex® Red Phospholipase D Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796
Fluorescent Substrates for Phospholipase D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797
EnzChek® Lipase Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797
Anti-Phosphoinositide Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797
Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797
BODIPY® Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 798
NBD Sphingolipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 799
Amplex® Red Sphingomyelinase Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 799
ADIFAB Reagent: A Unique Free Fatty Acid Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800
Data Table 17.4 Probes for Lipid Metabolism and Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 801
Product List 17.4 Probes for Lipid Metabolism and Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 802

The Molecular
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™
to Fluorescent
Fluorescent Probes
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andLabeling
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Technologies
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Chapter 17 — Probes for Signal Transduction Section 17.1 Introduction to Signal Transduction

17.1 Introduction to Signal Transduction

Cells respond to their environment through a complex and We offer several important reagents for studying signal transduction
interdependent series of signal transduction pathways that frequently mechanisms, including Ca 2+ regulation and second messenger
begin at the cell membrane. Many cellular receptors are transmembrane activities. This chapter focuses on probes for events occurring down-
proteins with extracellular domains that selectively bind ligands. In stream from the receptor–ligand interaction. These products comple-
response to ligand binding, the receptor’s cytoplasmic domain may ment the probes for receptors and ion channels in Chapter 16, as well
change conformation and transmit the signal across the membrane, as the many ion indicators discussed in Chapter 19, Chapter 20 and
or individual receptors may aggregate and interact with other membrane Chapter 21. Chapter 18 describes our selection of probes for nitric ox-
proteins in order to generate a response. Transmembrane signals trigger ide research—including nitric oxide donors, nitric oxide synthase in-
a cascade of events in the cell, which can include changes in intracel- hibitors and reagents for nitrite detection—as well as for other reactive
lular Ca 2+ levels, enzymatic activity and gene expression (Figure 17.1.1). oxygen species.

Adenylate cyclase–linked
G-protein–coupled receptors Ion channel–linked
G-protein–coupled receptors
RAC/Gi Phospholipase C–linked
G-protein–coupled receptors RION
5-HT1A
RAC/Gs 5-HT1B RPLC 5-HT1A (K+)
Ligand-gated ion channels
5-HT1D 5-HT1C (Cl–)
5-HT1A αt-Adrenergic 5-HT1A (transfected cells) 5-HT2 (Cl–)
RLG/ION
5-HT4 Muscarinic 5-HT1C (transfected cells) β-Adrenergic (Ca2+, Na+)
β-Adrenergic D2 Dopaminergic 5-HT2 (transfected cells) α2-Adrenergic (Ca2+, K+) 5-HT3 (Na+, K+)
D4 Dopaminergic A1 Adenosine α1-Adrenergic Muscarinic (K+, Ca2+) Nicotinic (Na+)
A2 Adenosine Opioid Muscarinic D2 Dopaminergic (Ca2+, K+) GABAA (Cl–)
VIP GABAB Metabotropic glutamate GABAB (K+) Ionotropic glutamate (Ca2+, Na+)

RPLC Ion
RAC RION channels RLG/ION
Gs
+ Gq
Gs Go
– AC PLC
Gi Gi
PTX K+ K+
PTX
ATP cAMP+Pi PIP2 DAG +IP3 Ca2+ Ca2+
Na+ Na+
Cl– Cl–

PKA PKC Ca2+

( ) ( ) ( )

Gs
( ) Cell proliferation.
( )
Gene expression (cAMP response elements), protein phosphorylation, Calcium can influence cell
changes in process outgrowth, secretion of growth factors from glia. proliferation, neurite elongation,
gene expression and cell viability.
Gi Cell proliferation. Neurite elongation.
( ) ( )

Figure 17.1.1 Neurotransmitter receptors linked to second messengers mediating growth responses in neuronal and nonneuronal cells. Abbreviations: RAC/Gs = Receptors coupled
to G-proteins that stimulate adenylate cyclase (AC) activity, leading to cAMP formation and enhanced activity of protein kinase A (PKA). RAC/Gi = Receptors coupled to pertussis toxin
(PTX)–sensitive G-proteins that inhibit adenylate cyclase activity. RPLC = Receptors promoting the hydrolysis of phosphatidylinositol 4,5-diphosphate (PIP2) to inositol 1,4,5-triphosphate
(IP3), which increases intracellular Ca2+, and diacylglycerol (DAG), which activates protein kinase C (PKC). RION = Receptors indirectly promoting ion fluxes due to coupling to various
G-proteins. RLG/ION = Receptors that promote ion fluxes directly because they are structurally linked to ion channels (members of the superfamily of ligand-gated ion channel recep-
tors). Stimulation of proliferation is most often associated with activation of G-proteins negatively coupled to adenylate cyclase (Gi), or positively coupled to phospholipase C (Gq) or to
pertussis toxin–sensitive pathways (Go, Gi). In contrast, activation of neurotransmitter receptors positively coupled to cAMP usually inhibits cell proliferation and causes changes in cell
shape indicative of differentiation. Reprinted and modified with permission from J.M. Lauder and Trends Neurosci (1993) 16:233. Learn more about gene specific products for signaling
pathways at [Link]/handbook/pathways.

The
TheMolecular
MolecularProbes Handbook:
Probes®
™
A Guide
Handbook: to Fluorescent
A Guide Probes
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Probes Labeling
and Technologies
Labeling Technologies
IMPORTANT NOTICE:described
The products described
manualinare
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manual are
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or moreUse
Limited Use Label License(s).
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on on
IMPORTANT NOTICE : The products in this one or more Label License(s). to the Appendix
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. 775
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 Chapter 17 — Probes for Signal Transduction Section 17.2 Calcium Regulation

17.2 Calcium Regulation

Intracellular Ca 2+ levels modulate a multitude of vital cellular pro- Fluorescent Heparin
cesses—including gene expression, cell viability, cell proliferation, cell Fluorescein-labeled heparin (H7482) is a useful tool for studying
motility, cell shape and volume regulation—thereby playing a key role binding of this mucopolysaccharide in cells and tissues.7,8 In addition
in regulating cell responses to external signals. These dynamic changes to its well-known anticoagulant activity,9 heparin binds to the Ins
in Ca 2+ levels are regulated by ligand-gated and G-protein–coupled ion 1,4,5-P3 receptor and inhibits the biological cascade of events mediated
channels in the plasma membrane and by mobilization of Ca 2+ from by Ins 1,4,5-P3.10 Heparin also binds to thrombin 11 and Alzheimer’s tau
intracellular stores. The generation of cytosolic Ca 2+ spikes and oscilla- protein,12 as well as to blood vessel–associated proteins such as laminin
tions typically involves the coordinated release and uptake of Ca 2+ from and fibronectin.13 Fluorescence polarization assays using fluorescein-
these stores, mediated by intracellular Ca 2+ channels and their response labeled heparin as a tracer provide quantitative assessments of these
to several second messengers such as Ca 2+ itself, cyclic ADP ribose and binding interactions.14 Fluorescein-labeled heparin has also been used
inositol triphosphate.1–3 to assess the efficacy of transdermal delivery of heparin by pulsed cur-
This section includes several Molecular Probes® reagents for rent iontophoresis as a potential alternative to conventional subcutane-
studying Ca2+ regulation in live cells. Fluorescent nucleotides, including ous injections.15
analogs of ATP, ADP, AMP, GTP, and GDP, are described in Section
17.3. Our GTP analogs may be particularly useful in the assay of
G-protein–coupled receptors. Section 17.4 discusses several selective Caged Ca2+ and Caged Ca2+ Chelators
phosopholipase substrates, as well as labeled ceramide and sphingo-
myelin probes. Caged ions and caged chelators can be used to influence the ionic
composition of both solutions and cells, particularly for ions such as
Ca 2+ that are present at low concentrations. The properties and uses of
Inositol Triphosphate Pathway caged probes are described in Section 5.3.

D-myo-1,4,5-Inositol Triphosphate and NP-EGTA: A Caged Ca2+ Reagent

Caged D-myo-1,4,5-Inositol Triphosphate Developed by Ellis-Davies and Kaplan, the photolabile chelator o-
We offer the potassium salt of D-myo-inositol 1,4,5-triphosphate nitrophenyl EGTA (NP-EGTA) exhibits a high selectivity for Ca 2+, a
(Ins 1,4,5-P3, I3716) for researchers investigating inositol triphosphate– dramatic 12,500-fold decrease in affinity for Ca 2+ upon UV illumination
dependent Ca2+ mobilization and signal transduction mechanisms.1 (its Kd increases from 80 nM to >1 mM) and a high photochemical
Cytoplasmic Ins 1,4,5-P3 is a potent intracellular second messenger that quantum yield 16,17 (~0.2). Furthermore, with a Kd for Mg2+ of 9 mM,
induces Ca2+ release from membrane-bound stores in many tissues. NP-caged EGTA does not perturb physiological levels of Mg2+. We of-
NPE-caged Ins 1,4,5-P3 can be used to generate rapid and precisely fer both the potassium salt (N6802) and the acetoxymethyl (AM) ester
controlled release of Ins 1,4,5-P3 in intact cells and is widely employed (N6803) of NP-EGTA. The NP-EGTA salt can be complexed with Ca 2+
in studies of Ins 1,4,5-P3–mediated second-messenger pathways.4–6 Our to generate a caged calcium complex that will rapidly deliver Ca2+ upon
NPE-caged Ins 1,4,5-P3 (I23580) is a mixture of the physiologically in- photolysis (Figure 17.2.2). The cell-permeant AM ester of NP-EGTA
ert, singly esterified P4 and P5 esters (Figure 17.2.1) and does not contain does not bind Ca 2+ unless the AM esters are removed. It can poten-
the somewhat physiologically active P1 ester. NPE-caged Ins 1,4,5-P3 tially serve as a photolabile buffer in cells because, once converted to
exhibits essentially no biological activity prior to photolytic release of NP-EGTA by intracellular esterases, it will bind Ca 2+ with high affinity
the biologically active Ins 1,4,5-P3. until photolyzed with UV light. NP-EGTA has been used to measure
the calcium buffering capacity of cells.18

_ _ _ _
Photocleavage OOC COO OOC COO
2+
N Ca N
O O

NO
2
Figure 17.2.1 D-myo-inositol 1,4,5-triphosphate, P4(5)-(1-(2-nitrophenyl)ethyl) ester, Figure 17.2.2 NP-EGTA (N6802) complexed with Ca2+. Upon illumination, this complex is
tris(triethylammonium) salt (NPE-caged Ins 1,4,5-P3) (I23580). cleaved to yield free Ca2+ and two iminodiacetic acid photoproducts. The affinity of the pho-
toproducts for Ca2+ is ~12,500-fold lower than that of NP-EGTA.

The
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Chapter 17 — Probes for Signal Transduction Section 17.2 Calcium Regulation

DMNP-EDTA: A Caged Ca2+ Reagent Other Probes for Calcium Regulation

The first caged Ca 2+ reagent described by Ellis-Davies and
Kaplan was 1-(4,5-dimethoxy-2-nitrophenyl) EDTA (DMNP-EDTA, Thapsigargin and Fluorescent Thapsigargin
D6814), which they named DM-Nitrophen™ 19,20 (now a trademark of Thapsigargin is a naturally occurring sesquiterpene lactone isolat-
Calbiochem-Novabiochem Corp.). Because its structure better resem- ed from the umbelliferous plant Thapsia garganica.32 This tumor pro-
bles that of EDTA than EGTA, we named it as a caged EDTA derivative moter releases Ca 2+ from intracellular stores by specifically inhibiting
(Figure 17.2.3). Upon illumination, DMNP-EDTA’s Kd for Ca 2+ increas- the sarcoplasmic reticulum Ca 2+-ATPase 33,34 (SERCA); it does not di-
es from 5 nM to 3 mM. Thus, photolysis of DMNP-EDTA complexed rectly affect plasma membrane Ca 2+-ATPases, Ins 1,4,5-P3 production
with Ca 2+ results in a pulse of free Ca 2+. Furthermore, DMNP-EDTA or protein kinase C activity.35,36
has significantly higher affinity for Mg2+ (Kd = 2.5 µM) than does NP- Thapsigargin is available in 1 mg units (T7458) and specially pack-
EGTA 19 (Kd = 9 mM). The photolysis product’s Kd for Mg2+ is ~3 mM, aged in 20 vials containing 50 µg each (T7459). We have also prepared
making DMNP-EDTA an effective caged Mg2+ source, in addition to the green-fluorescent BODIPY® FL thapsigargin (B7487, Figure 17.2.5)
its applications for photolytic Ca 2+ release.21,22 Photorelease of Ca 2+ has and red-fluorescent BODIPY® TR-X thapsigargin (B13800, Figure
been shown to occur in <180 microseconds, with even faster photore- 17.2.6). BODIPY® FL thapsigargin has proven useful for imaging the intra-
lease of Mg2+.23 Two reviews by Ellis-Davies discuss the uses and limita- cellular localization of thapsigargin during store-operated calcium entry
tions of DMNP-EDTA.24,25 (SOCE) 37 and for imaging SERCA depletion in injured sensory neurons.38

Diazo-2: A Photoactivatable Ca2+ Knockdown Reagent Luminescent Calcium Analog

In contrast to NP-EGTA and DMNP-EDTA, diazo-2 (D3034) is a The trivalent lanthanide terbium (III), which is supplied as its chlo-
photoactivatable Ca 2+ scavenger. Diazo-2 (Figure 17.2.4), which was in- ride salt (T1247), is a luminescent analog of Ca 2+ that can be used to
troduced by Adams, Kao and Tsien,26,27 is a relatively weak chelator (Kd study structure–function relationships in Ca 2+-binding proteins such
for Ca 2+ = 2.2 µM). Following flash photolysis at ~360 nm, however, cy- as calmodulin, oncomodulin, lactalbumin and ATPases.39–41 The long-
tosolic free Ca 2+ rapidly binds to the diazo-2 photolysis product, which lived luminescence of Tb3+ has also been use to probe Ca 2+-binding sites
has a high affinity for Ca 2+ (Kd = 73 nM). Microinjecting a relatively low of alkaline phosphatase,42 glutamine synthetase,43 integrins, 39 protein
concentration of fluo-3, fluo-4, or one of the Calcium Green™ or Oregon kinase C 44 and ryanodine-sensitive Ca 2+ channels.45 Tb3+ reportedly
Green® 488 BAPTA indicators (Section 19.3), along with a known quan- binds most strongly to the I and II sites of calmodulin.46
tity of diazo-2, permits measurement of the extent of depletion of cyto-
solic Ca 2+ following photolysis.27–29 Intracellular loading of NP-EGTA,
DMNP-EDTA and diazo-2 is best accomplished by patch pipette infu-
sion with the carboxylate salt form of the caged compound added to the
internal pipette solution at 1–10 mM. These reagents are increasingly
being applied in vivo for controlled intervention in calcium-regulated
fundamental processes in neurobiology 30 and developmental biology.31

_ _
Photocleavage OOC COO
_
N 2+ COO
Ca
CH O N _
3
COO
CH O NO
3 2
Figure 17.2.3 DMNP-EDTA (D6814) complexed with Ca2+. Upon illumination, this complex Figure 17.2.5 BODIPY® FL thapsigargin (B7487).
is cleaved to yield free Ca2+ and two iminodiacetic acid photoproducts. The affinity of the
photoproducts for Ca2+ is ~600,000-fold lower than that of DMNP-EDTA.

Figure 17.2.4 Diazo-2, tetrapotassium salt (D3034). Figure 17.2.6 BODIPY® TR-X thapsigargin (B13800).

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 Chapter 17 — Probes for Signal Transduction Section 17.2 Calcium Regulation

REFERENCES
1. Biochim Biophys Acta (2009) 1793:933; 2. Cell (2007) 131:1047; 3. Nat Cell Biol (2009) Biophys Acta (1990) 1035:378; 27. J Am Chem Soc (1989) 111:7957; 28. Nature (1994)
11:669; 4. Neuron (2007) 54:611; 5. J Physiol (1995) 487:343; 6. Neuron (1995) 15:755; 371:603; 29. Biophys J (1993) 65:2537; 30. Science (2009) 325:207; 31. Dev Growth Differ
7. Biochem Biophys Res Commun (2006) 348:850; 8. Chem Biol (2004) 11:487; 9. J Biol (2009) 51:617; 32. Acta Pharm Suec (1978) 15:133; 33. J Biol Chem (1998) 273:12994;
Chem (1992) 267:8857; 10. Biochem J (1994) 302:155; 11. J Biol Chem (1998) 273:34730; 34. J Biol Chem (1995) 270:11731; 35. Proc Natl Acad Sci U S A (1990) 87:2466;
12. Biochemistry (2006) 45:6446; 13. J Biol Chem (1995) 270:18558; 14. J Biol Chem 36. J Biol Chem (1989) 264:12266; 37. J Biol Chem (2007) 282:12176; 38. Anesthesiology
(2008) 283:19389; 15. Pharm Res (2006) 23:114; 16. J Biol Chem (1995) 270:23966; (2009) 111:393; 39. Biochemistry (1994) 33:12238; 40. J Biol Chem (1992) 267:13340;
17. Proc Natl Acad Sci U S A (1994) 91:187; 18. Biochem Biophys Res Commun (1998) 41. Photochem Photobiol (1987) 46:1067; 42. J Photochem Photobiol B (1992) 13:289;
250:786; 19. Proc Natl Acad Sci U S A (1988) 85:6571; 20. Science (1988) 241:842; 43. Biochemistry (1991) 30:3417; 44. J Biol Chem (1988) 263:4223; 45. J Biol Chem
21. Methods Cell Biol (1994) 40:31; 22. Neuron (1993) 10:21; 23. Biochemistry (1992) (1994) 269:24864; 46. Biochem Biophys Res Commun (1986) 138:1243.
31:8856; 24. Chem Rev (2008) 108:1603; 25. Nat Methods (2007) 4:619; 26. Biochim

DATA TABLE 17.2 CALCIUM REGULATION

Cat. No. MW Storage Soluble Abs EC Em Solvent Notes
B7487 854.75 FF,D,L DMSO 503 85,000 511 MeOH
B13800 1100.04 FF,D,L DMSO 589 62,000 616 MeOH
D3034 710.86 F,D,LL pH >6 369 18,000 none pH 7.2 1, 3, 4
D6814 473.39 D,LL DMSO 348 4200 none pH 7.2 1, 4, 5
H7482 ~18,000 FF,D,L H2O 493 ND 514 pH 8 6, 7
I3716 648.64 F,D H2O <250 none
I23580 872.82 FF,D,LL H2O 264 4200 none H2O 1, 2, 8
N6802 653.81 FF,D,LL pH >6 260 3500 none pH 7.2 1, 2, 4, 9
N6803 789.70 FF,D,LL DMSO 250 4200 none MeCN 10, 11
T1247 373.38 D H2O 270 4700 545 H2O 12, 13
T7458 650.76 F,D DMSO, EtOH <300 none
T7459 650.76 F,D DMSO, EtOH <300 none
For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.
Notes
1. All photoactivatable probes are sensitive to light. They should be protected from illumination except when photolysis is intended.
2. This compound has weaker visible absorption at >300 nm but no discernible absorption peaks in this region.
3. The Ca2+ dissociation constant of diazo-2 is 2200 nM before photolysis and 73 nM after ultraviolet photolysis. The absorption spectrum of the photolysis product is similar to that of BAPTA. (J Am
Chem Soc (1989) 111:7957)
4. Abs and EC values determined in Ca2+-free solution (100 mM KCl, 10 mM EGTA, 10 mM MOPS, pH 7.2).
5. Kd (Ca2+) increases from 5 nM to 3 mM after ultraviolet photolysis. Kd values determined in 130 mM KCl, 10 mM HEPES, pH 7.1. (Proc Natl Acad Sci U S A (1988) 85:6571)
6. ND = not determined.
7. This product is a multiply labeled bioconjugate. The number of labels per conjugate is indicated on the vial.
8. Ultraviolet photolysis of I23580 generates I3716.
9. Kd (Ca2+) increases from 80 nM to 1 mM after ultraviolet photolysis. Kd values determined in 100 mM KCl, 40 mM HEPES, pH 7.2. (Proc Natl Acad Sci U S A (1994) 91:187)
10. This product is intrinsically a liquid or an oil at room temperature.
11. N6803 is converted to N6802 via hydrolysis of its acetoxymethyl ester (AM) groups.
12. Absorption and luminescence of T1247 are extremely weak unless it is chelated. Data are for dipicolinic acid (DPA) chelate. The luminescence spectrum has secondary peak at 490 nm.
13. MW is for the hydrated form of this product.

PRODUCT LIST 17.2 CALCIUM REGULATION

Cat. No. Product Quantity
A7621 8-amino-cyclic adenosine 5’-diphosphate ribose (8-amino-cADP-ribose) 10 µg
B7487 BODIPY® FL thapsigargin 100 µg
B13800 BODIPY® TR-X thapsigargin 5 µg
C7074 cyclic adenosine 5’-diphosphate ribose, 1-(1-(2-nitrophenyl)ethyl) ester (NPE-caged cADP-ribose) *mixed isomers* 50 µg
D3034 diazo-2, tetrapotassium salt *cell impermeant* 1 mg
D6814 1-(4,5-dimethoxy-2-nitrophenyl)-1,2-diaminoethane-N,N,N’,N’-tetraacetic acid (DMNP-EDTA) *cell impermeant* 5 mg
H7482 heparin, fluorescein conjugate 1 mg
I3716 D-myo-inositol 1,4,5-triphosphate, hexapotassium salt (Ins 1,4,5-P3) 1 mg
I23580 D-myo-inositol 1,4,5-triphosphate, P4(5)-(1-(2-nitrophenyl)ethyl) ester, tris(triethylammonium) salt (NPE-caged Ins 1,4,5-P3) 25 µg
N6803 o-nitrophenyl EGTA, AM (NP-EGTA, AM) *cell permeant* *special packaging* 20 x 50 µg
N6802 o-nitrophenyl EGTA, tetrapotassium salt (NP-EGTA) *cell impermeant* 1 mg
T1247 terbium(III) chloride, hexahydrate 1g
T7458 thapsigargin 1 mg
T7459 thapsigargin *special packaging* 20 x 50 µg

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page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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Chapter 17 — Probes for Signal Transduction Section 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins

17.3 Probes for Protein Kinases, Protein Phosphatases

and Nucleotide-Binding Proteins
The cascade of cellular events in response to an internal signal or Protein Kinase Probes and Assays
environmental stimulus requires a diversity of molecular participants,
ranging from ions to enzymes. Signal transduction pathways frequent- Protein kinases are critical players in signal transduction path-
ly activate specific protein kinases, leading to the phosphorylation of ways. The fluorometric assay of kinases, however, is not straightforward
particular cellular proteins and subsequent initiation of a multitude of because ATP-dependent phosphorylation of a fluorescent peptide sub-
cellular responses. Binding and hydrolysis of nucleotides plays a ma- strate does not directly lead to appreciable changes in the fluorescence
jor role in these activities, and our nucleotide analogs and assays for of the product.1,2 We provide an extensive range of assays for protein
phosphate-producing enzymes are important tools for signal transduc- kinases that utilize a variety of strategies to detect phosphorylation of
tion research and high-throughput screening of compounds that affect peptide and protein substrates (Table 17.1).
signal transduction.
We offer a selection of native and modified biomolecules to aid the Antibody Beacon™ Tyrosine Kinase Assay Kit
researcher in dissecting this highly complex branch of the signal trans- The Antibody Beacon™ Tyrosine Kinase Assay Kit (A35725) provides
duction process. In addition to the probes below, we have developed the a homogeneous solution assay for measuring the activity of tyrosine
PiPer™ and EnzChek® assay kits for quantitation of inorganic phosphate kinases and the effectiveness of potential inhibitors and modulators.3
and pyrophosphate that are extremely useful for following hydrolysis The key to this tyrosine kinase assay is a small-molecule tracer ligand
of nucleotides by various enzymes and of phosphate esters by protein labeled with our bright green-fluorescent Oregon Green® 488 dye. When
phosphatases. These kits and other kits to measure ATP by chemilumi- an anti-phosphotyrosine antibody binds this tracer ligand to form the
nescence and protein phosphatase activity are described in Section 10.3. Antibody Beacon™ detection complex, the fluorescence of the Oregon

Table 17.1 Invitrogen kinase assay platforms.

Assay Principle References
Adapta® Universal Kinase Assay In the absence of an inhibitor, ADP formed by a kinase reaction will displace an Alexa Fluor® 647 dye–labeled
ADPtracer from an Eu3+-labeled anti-ADP antibody, resulting in a decrease in the TR-FRET* signal. In the presence of
an inhibitor, the amount of ADP formed by the kinase reaction is reduced, and the resulting intact antibody–tracer
interaction produces a high TR-FRET signal.
Antibody Beacon™ Tyrosine Kinase Assay Peptide substrate phosphorylation is detected via competitive displacement of an Oregon Green® 488 dye–labeled 1
ligand from a phosphospecific antibody.
LanthaScreen® Kinase Activity Assays A terbium (Tb3+)– or europium (Eu3+)–labeled phosphospecific antibody binds the phosphorylated fluorescein- or 2, 3
Alexa Fluor® 647 dye–labeled peptide substrate, resulting in an increase in the TR-FRET signal.
LanthaScreen® Eu Kinase Binding Assay Binding of an Alexa Fluor® 647 tracer to a kinase is detected by addition of a Eu3+-labeled anti–epitope tag 4
antibody. Binding of the tracer and antibody to a kinase results in a high FRET signal, whereas displacement of the
tracer by a kinase inhibitor results in a loss of FRET signal.
LanthaScreen® Cellular Assays Detection of phosphorylation or other protein modification event is measured on a TR-FRET–compatible plate 5, 6
reader. Little or no TR-FRET is observed with unstimulated or inhibited cells, whereas stimulated cell samples display
high TR-FRET.
NDP Sensor Protein This fluorescent ADP/ATP biosensor consists of a recombinant bacterial nucleoside diphosphate kinase site- 7
specifically labeled with an environment-sensitive coumarin dye.
Omnia® Kinase Assay Fluorescence enhancement of N- or C-terminal 8-hydroxyquinoline fluorophore (Sox) upon chelation of Mg2+ is 8, 9
coupled to phosphorylation of a peptide substrate at an adjacent Ser, Thr or Tyr residue.
Z´-LYTE® Kinase Assay Phosphorylation-dependent protease susceptibility of a double-labeled peptide substrate is detected using FRET. 10
CellSensor® Cell Lines CellSensor® assays measure pathway-driven activation of transcription factors using GeneBLAzer® β-lactamase 11–13
reporter technology. Minimal amounts of β-lactamase are expressed in untreated cells or cells treated with a
pathway-specific inhibitor. Stimulation of the pathway with a ligand or with a constitutively active mutation in a
pathway component leads to activation of downstream transcription factor(s), resulting in β-lactamase reporter
gene expression. Cells are loaded with a cell-permeable β-lactamase substrate, and β-lactamase reporter activity is
measured on a fluorescence plate reader.
1. Free Radic Biol Med (2009) 47:983; 2. J Biomol Screen (2009) 14:121; 3. Anal Biochem (2006) 356:108; 4. J Biomol Screen (2009) 14:924; 5. J Biomol Screen (2009) 14:121; 6. Anal Biochem (2008)
372:189; 7. Biochemistry (2001) 40:5087; 8. Anal Biochem (2006) 352:198; 9. J Am Chem Soc (2003) 125:14248; 10. Assay Drug Dev Technol (2002) 1:9; 11. Current Chemical Genomics (2009) 3:1;
12. Mol Biosyst (2009) 5:1039; 13. Mol Cancer (2009) 8:117.
* TR-FRET = Time-resolved fluorescence resonance energy transfer. For further information on these assay technologies and other kinase biology products and services, visit
[Link]/handbook/kinase.

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 Chapter 17 — Probes for Signal Transduction Section 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins

Green® 488 dye is efficiently quenched. In the presence of a phosphoty- • Compatibility. The anti-phosphotyrosine antibody provided in the
rosine-containing peptide, however, this Antibody Beacon™ detection Antibody Beacon™ Tyrosine Kinase Assay Kit is specific for phos-
complex is rapidly disrupted, releasing the tracer ligand and relieving photyrosine residues; assay components such as ATP (up to 1 mM)
its antibody-induced quenching (Figure 17.3.1). Upon its displacement and reducing agents such as dithiothreitol (DTT, up to 1 mM) do not
by a phosphotyrosine residue, the Oregon Green® 488 dye–labeled tracer interfere with this assay. This anti-phosphotyrosine antibody was se-
ligand exhibits an approximately 4-fold fluorescence enhancement, en- lected from among several clones to produce the greatest fluorescence
abling the detection of as little as 50 nM phosphotyrosine-containing enhancement by the kinase-phosphorylated product.
peptide with excellent signal-to-background discrimination. Key ben- • Reliability. This tyrosine kinase assay has a broad signal window,4
efits of the Antibody Beacon™ Tyrosine Kinase Assay Kit include: indicated by a Z´ factor of >0.85.

• Real-time measurements. Unlike many other commercially avail- The Antibody Beacon™ Tyrosine Kinase Assay Kit comes with all
able tyrosine kinase assays, the Antibody Beacon™ Tyrosine Kinase the reagents needed to perform this assay, including:
Assay Kit permits real-time monitoring of kinase activity (Figure
17.3.2). Not only is the Antibody Beacon™ detection complex rap- • Oregon Green® 488 dye–labeled tracer ligand
idly dissociated in the presence of phosphotyrosine residues, but • Anti-phosphotyrosine antibody
the assay components have been designed to be simultaneously • Concentrated tyrosine kinase reaction buffer
combined, reducing any delay in the measurements. • Two generic tyrosine kinase substrate solutions: a poly(Glu:Tyr)
• Simple detection protocol. Tyrosine kinase activity is measured by solution and a poly(Glu:Ala:Tyr) solution
a simple increase in fluorescence intensity; no special equipment, • Dithiothreitol (DTT)
additional reagents, or extra steps are required. This assay is readily • Adenosine triphosphate (ATP)
compatible with any fluorescence microplate reader. • Phosphotyrosine-containing peptide, phospho-pp60 c-src (521–
• Use of natural substrates. The Antibody Beacon™ tyrosine kinase 533), for use as a reference
assay utilizes unlabeled peptide or protein substrates, is compatible • Detailed protocols
wth substrates that are pre-phosphorylated at serine or threonine
(but not at tyrosine) residues and is applicable to the assay of a wide Each kit provides sufficient reagents to perform ~400 assays using
variety of kinases. a 50 µL assay volume in a fluorescence microplate reader.

Fluorescent Polymyxin B Analogs

Polymyxin B is a cyclic polycationic peptide antibiotic (Figure
17.3.3) that binds to lipopolysaccharides and anionic lipids.5 Polymyxin
A I Y A E
A I pY A E
B is also a selective inhibitor of protein kinase C, with an IC50 of
~35 µM,6–9 as well as a potent inhibitor of calmodulin, with an IC50 of
Substrate

80 nM in the presence of 500 µM Ca 2+.10 Our fluorescent polymyxin

Tyrosine kinase
B analogs include those of the green-fluorescent BODIPY® FL 11 and
Oregon Green® 514 fluorophores (P13235, P13236), as well as the ultra-
Ligand-antibody
detection complex
ATP ADP Phosphorylated substrate
and displaced ligand
violet light–excitable dansyl polymyxin 5 (P13238).

Figure 17.3.1 Reaction scheme for the tyrosine kinase assay used in the Antibody Beacon™
Tyrosine Kinase Assay Kit (A35725). The unlabeled natural substrate (AIYAE) is phosphory-
Hypericin
lated by the tyrosine kinase to AIY(P)AE, which displaces the quenched Oregon Green® 488 Hypericin (H7476, Figure 17.3.4), an anthraquinone derivative isolat-
dye–labeled peptide from the anti-phosphotyrosine antibody, resulting in a large increase in ed from plants of the genus Hypericum,12,13 is a potent, selective inhibitor
its fluorescence that is proportional to the amount of AIY(P)AE formed in the reaction. of PKC (IC50 = 1.7 µg/mL = 3.4 µM) useful for probing and manipulating
PKC in live cells.14 Hypericin has a variety of pharmacological properties,
from antibacterial and antineoplastic activities to antiviral activities 15–18
and induction of apoptosis.19 Hypericin is also a potent photosensitizer,
800
with a quantum yield of 0.75 for the generation of singlet oxygen.20
600 Koff = 0.14 sec-1
Fluorescence

400

200 NH3 NH3

O O
O O H H
0 Addition of phosphotyrosine- H H N N
containing peptide N N N
N N H
H H O
0 20 40 60 80 100 120 140 O O NH3
OH HO
O HN O
Time (seconds) H
HN N
N
Figure 17.3.2 Real-time detection capability of the Antibody Beacon™ Tyrosine Kinase Assay H
Kit (A35725). Fluorescence of the Antibody Beacon™ detection complex in tyrosine kinase assay O O
NH3
buffer was monitored over time. After ~15 seconds, an excess of phosphotyrosine-containing
NH3
peptide was added to the Antibody Beacon™ detection complex and the off-rate was calculated. Figure 17.3.3 Polymyxin B.

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toFluorescent
™
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Fluorescent Probes andLabeling
LabelingTechnologies
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coveredare
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one or more Limited Use Label License(s). Please refer to thePlease
Appendix onto
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Chapter 17 — Probes for Signal Transduction Section 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins

Protein Phosphatase Assay Kits

RediPlate™ 96 EnzChek® Tyrosine Phosphatase Assay Kits
Protein tyrosine phosphatases (PTP) represent a large family of enzymes that play a very
important role in intra- and intercellular signaling. PTPs work antagonistically with protein
tyrosine kinases to regulate signal transduction pathways in response to a variety of signals,
including hormones and mitogens.3 Our RediPlate™ 96 EnzChek® Tyrosine Phosphatase Assay Figure 17.3.4 Hypericin (H7476).
Kit (R22067) provides researchers with a sensitive and convenient method to monitor PTP and
screen PTP inhibitors in a variety of research areas.21–24
The EnzChek® tyrosine phosphatase assay is based on 6,8-difluoro-4-methylumbellifer- 12

yl phosphate 25 (DiFMUP, D6567, D22065; Section 10.3). Unlike other end-point tyrosine 10
phosphatase assay kits, the EnzChek® tyrosine phosphatase assay is continuous, allowing

Fluorescence
8
researchers to easily measure fluorescence at various time points in order to follow the kinetics
of the reaction. Furthermore, the assay is not affected by free phosphate and is compatible 6

with most nonionic detergents, resulting in minimal sample processing before analysis. Most 4

importantly, each assay well contains inhibitors to help ensure that the assay is selective for 2
tyrosine phosphatases; other phosphatases, including serine/threonine phosphatases, will not
hydrolyze DiFMUP under our assay conditions (Figure 17.3.5). Unlike phosphopeptide-based 0
0 5 10 15 20 25 30 35 40 45 50
assays, this DiFMUP-based assay can be used to monitor a variety of tyrosine phosphatases, Time (min)
including PTP-1B and CD-45 (Figure 17.3.5). Tyrosine phosphatase inhibitors can be evalu- Symbol Enzyme (Class) Enzyme Units*
ated quantitatively in the assay for their effect on tyrosine phosphatase activity. CD-45 (tyrosine phosphatase) 1 U/mL
PTP-1B (tyrosine phosphatase) 1 mU/mL
Each RediPlate™ 96 EnzChek® Tyrosine Phosphatase Assay Kit (R22067) includes: PTPase (tyrosine phosphatase) 1 U/mL
Acid phosphatase 1 U/mL
Alkaline phosphatase 1 U/mL
• One RediPlate™ 96 EnzChek® tyrosine phosphatase assay 96-well microplate PP2A (ser/thr phosphatase)† 1 U/mL
• Reaction buffer PP1 (ser/thr phosphatase)† 1 U/mL

• Detailed assay protocols PP-2B (ser/thr phosphatase)† 500 U/mL

* Enzyme unit (U) definitions are standard definitions for each enzyme.
† Serine theonine phosphatase.

RediPlate™ 96 EnzChek® Serine/Threonine Phosphatase Assay Kit Figure 17.3.5 Specificity of the RediPlate™ 96 EnzChek®
The majority of protein phosphorylation occurs on serine and threonine residues, with Tyrosine Phosphatase Assay Kit (R22067). The phosphatases
<0.01–0.05% on tyrosine residues. Serine/threonine phosphatases represent a large family of listed in the tables were applied to a RediPlate™ 96
EnzChek® tyrosine phosphatase assay microplate. At the
enzymes that have been implicated in the regulation of metabolism, transcription, translation,
indicated time points, the fluorescence was measured in
differentiation, cell cycle, cytoskeletal dynamics, oncogenesis and signal transduction. The a fluorescence microplate reader using excitation at 355 ±
RediPlate™ 96 EnzChek® Serine/Threonine Phosphatase Assay Kit (R33700) provides a fast, sim- 20 nm and emission at 460 ± 12.5 nm.
ple and direct fluorescence-based assay for detecting serine/threonine phosphatases and their
corresponding modulators and inhibitors 25 (Figure 17.3.6).
120,000
As with the RediPlate™ 96 EnzChek® Tyrosine Phosphatase Kit, the substrate incorporated
in the RediPlate™ 96 EnzChek® Serine/Threonine Phosphatase Assay Kit is DiFMUP. Inhibitors 100,000

are included in each assay well to help ensure that the assay is selective for serine/threonine
Fluorescence

80,000

phosphatases; under the prescribed assay conditions, other phosphatases, including tyrosine
60,000
phosphatases, do not significantly react with the substrate (Figure 17.3.7). Furthermore, unlike
phosphopeptide-based assays, this DiFMUP-based assay can be used to monitor a variety of ser- 40,000

ine/threonine phosphatases including PP-1, PP-2A and PP-2B (Figure 17.3.7). Serine/threonine 20,000

phosphatase inhibitors can be evaluated quantitatively in the assay for their effect on serine/
0
0 0.2 0.4 0.6
Enzyme (U/ml)

F Symbol Enzyme (Class)

O F
HO O O PP-2A (Ser/Thr phosphatase)
HO P O O O
PP-1 (Ser/Thr phosphatase)
OH
PP-2B (Ser/Thr phosphatase)
F
F
Alkaline phosphatase
CH
CH 3
3 Acid phosphatase
DiFMUP DiFMU LAR (tyrosine phosphatase)
(nonfluorescent) (fluorescent)
Figure 17.3.7 Specificity of the RediPlate™ 96 EnzChek®
Serine/Threonine Phosphatase Assay Kit (R33700) for serine/
threonine phosphatases. The phosphatases listed in the tables
were applied at the indicated concentrations to a RediPlate™
Incubate
Add reaction buffer, then 20–30 minutes Measure fluorescence 96 EnzChek® serine/threonine phosphatase assay micro-
add sample containing PTPase (excitation/emission ~355/455 nm) plate. Reactions were incubated at 37°C. After 1 hour, fluores-
cence was measured in a fluorescence microplate reader us-
Figure 17.3.6 Schematic diagram of the method used in the RediPlate™ EnzChek® Phosphatase Assay Kits (R22067, R33700). ing excitation at 355 ± 20 nm and emission at 460 ± 12.5 nm.

The
TheMolecular
MolecularProbes Handbook:
Probes®
™
A Guide
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A Guide Probes
to Fluorescent and
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and Technologies
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IMPORTANT NOTICE:described
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manualinare
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manual are
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 Chapter 17 — Probes for Signal Transduction Section 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins

threonine phosphatase activity (Figure 17.3.8). Additional advantages of this RediPlate™ assay
include compatibility with nonionic detergents and insensitivity to free phosphate, minimizing
sample processing before analysis.
Each RediPlate™ 96 EnzChek® Serine/Threonine Phosphatase Assay Kit includes:

• One RediPlate™ 96 EnzChek® serine/threonine phosphatase assay 96-well microplate

• Concentrated reaction buffer
• NiCl2
• MnCl2
• Dithiothreitol
• Detailed assay protocols

Figure 17.3.8 Detection of PP-2A inhibition by okadaic To ensure the integrity of the predispensed reagents, the 96-well microplate provided in both
acid using the RediPlate™ 96 EnzChek® Serine/Threonine RediPlate™ Protein Phosphatase Assay Kits is packaged in a resealable foil pouch and consists
Phosphatase Assay Kit (R33700). Each reaction contained
50 µM DiFMUP, 10 mU/mL PP-2A and the indicated concen- of twelve removable strips, each with eight wells (Figure 17.3.9). Eleven of the strips (88 wells)
tration (log scale) of okadaic acid in reaction buffer contain- are preloaded with the fluorogenic substrate DiFMUP; the remaining strip, marked with black
ing 50 mM Tris-HCl, 0.1 mM CaCl2, 1 mM NiCl2, 125 µg/mL tabs, contains a dilution series of the DiFMU reference standard for generating a standard curve.
bovine serum albumin (BSA) and 0.05% Tween® 20. Reactions
were incubated at 37°C. After 30 minutes, fluorescence was
measured in a fluorescence microplate reader using excita- Pro-Q® Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit
tion at 355 ± 20 nm and emission at 460 ± 12.5 nm. The Pro-Q® Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit (P33706) pro-
vides a method for selectively staining phosphoproteins or phosphopeptides on microarrays with-
out the use of antibodies or radioactivity. This kit permits direct detection of phosphate groups

NOTE 17.1
G-Proteins and GTP Analogs for Binding Studies
We prepare a wide variety of nucleotide analogs for protein-binding studies; their chemical and
spectral properties are described in Section 17.3. These include various fluorescent, photoaffinity
and caged versions of adenosine and guanosine triphosphates, diphosphates and cyclic monophos-
phates. The GTP analogs are among the most important probes for the study of G-proteins and G
Figure 17.3.9 A RediPlate™ 96 microplate. protein–coupled receptors (GPCR). Heterotrimeric guanine nucleotide–binding regulatory proteins
transmit a variety of receptor signals to modulate diverse cellular responses,1,2 including apoptosis.3
G-proteins are composed of α-, β- and γ-subunits. Upon receptor stimulus, the α-subunit
of the heterotrimeric G-proteins exchanges GDP for GTP and dissociates from the β-γ-subunit
complex. The GTP-bound G protein will interact with various second-messenger systems, either
inhibiting (Gi) or stimulating (Gs) their activity. Stimulatory G-proteins are permanently activated
by cholera toxin, inhibitory G-proteins by pertussis toxin. The α-subunit has a slow intrinsic rate of
GTP hydrolysis, and once the GTP is hydrolyzed it reassociates with the β-γ-subunit complex. The
GTP hydrolysis by G-proteins is regulated by interactions with GTPase-activating proteins, or
GAPs. There is a large family of GAPs for G-proteins known as regulators of G protein signaling
or, RGS proteins.4 G-proteins are turned off when the α-subunit hydrolyzes the GTP, either spon-
taneously or upon interaction with a GTPase-activating protein, permitting the heterotrimeric
α-β-γ-complex to reassociate.
The GAPs are a diverse group of monomeric GTPases, including ARF, Ran, Ras, Rab, Rac, Rho and
Sar, which play an important part in regulating many intracellular processes, such as cytoskeletal
organization and secretion. There is less diversity among the β- and γ-subunits, but they may have
direct activating effects in their own right. Most β- and γ-subunits are posttranslationally modified
by myristoylation or isoprenylation, which may alter their association with membranes.
Our fluorescent GTP analogs include:

• Blue-fluorescent MANT-GTP (M12415) and nonhydrolyzable MANT-GMPPNP (M22353)

• Green-fluorescent BODIPY® FL guanosine 5’-triphosphate (BODIPY® FL GTP, G12411)
• Red-fluorescent BODIPY® TR guanosine 5’-triphosphate (BODIPY® TR GTP, G22351)
• Nonhydrolyzable green-fluorescent BODIPY® FL GTP-γ-S thioester (G22183)

1. Annu Rev Biochem (2008) 77:1; 2. Physiol Rev (1999) 79:1373; 3. J Biol Chem (2000) 275:20726; 4. J Biol Chem
(1998) 273:1269.

The
TheMolecular
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
Guideto
™
toFluorescent
Fluorescent Probes
Probes and
and Labeling
LabelingTechnologies
Technologies
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
782 IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer tothe Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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Chapter 17 — Probes for Signal Transduction Section 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins

attached to tyrosine, serine or threonine residues in a microarray substrate⁄enhancer reagent generates sustained glow light emission that
environment and has been optimized for microarrays with acrylamide is measured 30 minutes after addition. Once the substrate⁄enhancer
gel surfaces. Each Pro-Q® Diamond Phosphoprotein/Phosphopeptide reaches the glow signal, the plate can be read for hours with little or
Microarray Stain Kit provides: no degradation of the signal, facilitating screening protocols in which
several plates are compared to each other. In addition, the assay exhibits
• Pro-Q® Diamond phosphoprotein/phosphopeptide microarray stain exceptionally low cross-reactivity with other adenosine-containing or
• Pro-Q® Diamond microarray destain solution cyclic nucleotides.
• Microarray staining gasket with seal tabs, 10 chambers The cAMP Chemiluminescent Immunoassay Kit (2-plate size, C10557;
• Slide holder tube, 20 tubes 10-plate size, C10558) is designed for the rapid and sensitive quantitation of
• Detailed protocols cAMP in extracts prepared from mammalian cells cultured in microwell
plates. Each kit provides all required reagents, including:
The Pro-Q® Diamond Phosphoprotein/Phosphopeptide Microarray
Stain Kit is ideal for identifying kinase targets in signal transduction • Alkaline phosphate conjugate of cAMP
pathways and for phosphoproteomics studies.26 • Anti-cAMP antibody
• cAMP standard
• CSPD® substrate and Sapphire™-II luminescence enhancer
Adenylate Cyclase Assays • Assay and lysis buffer
• Conjugate dilution buffer
3´,5´-Cyclic AMP (cAMP) is an important second messenger in many • Wash buffer
signal transduction pathways, linking activation of cell-surface mem- • Precoated microplates
brane receptors to intracellular responses, and, ultimately, to changes • Detailed protocols
in gene expression. cAMP is synthesized by plasma membrane–bound
adenylate cyclase, which is coupled to transmembrane receptors for hor- The cAMP Chemiluminescent Immunoassay Kit is designed for
mones, neurotransmitters and other signaling molecules by heterotri- quantitating cellular cAMP for functional assays of receptor activa-
meric G-proteins. Upon ligand binding, the intracellular receptor do- tion. It has been used with established cell lines for functional mea-
main of a G-protein–coupled receptor (GPCR) interacts with a G-protein, surements with endogenous receptors,29–32 with cell lines containing
which then dissociates and activates adenylate cyclase, resulting in an exogenously expressed ligand receptors, 33,34 with primary cells 35,36 and
increase in the concentration of intracellular cAMP. Subsequently, cAMP with tissues.37 It has also been used for receptor characterization, 38
activates cAMP-dependent protein kinases (protein kinase A), which orphan receptor ligand identification 39 and the characterization of
phosphorylate specific substrate proteins, including enzymes, structural novel chimeric receptors.40 In addition, this assay can be used for high-
proteins, transcription factors and ion channels. throughput screening assays 41 of compounds that stimulate or interfere
with these signal transduction pathways.
Adenylate Cyclase Probe: BODIPY® FL Forskolin
Forskolin, isolated from Coleus forskohlii, is a potent activator of
adenylate cyclase, the enzyme that catalyzes the formation of cAMP Nucleotide Analogs
from ATP. Green-fluorescent BODIPY® FL forskolin (B7469, Figure
17.3.10) has been used to visualize adenylyl cyclase internalization and Nucleotide analogs that serve as substrates or inhibitors of en-
subcellular distribution, 27 as well as for the pharmacological character- zymes, as well as nucleotide derivatives that selectively bind to regula-
ization of adenylyl cyclase catalytic subunits.28 tory sites of nucleotide-binding proteins, have been used as structural
and mechanistic probes for isolated proteins, reconstituted membrane-
cAMP Chemiluminescent Immunoassay Kit bound enzymes, organelles such as mitochondria, and tissues such as
The cAMP Chemiluminescent Immunoassay Kit enables ultrasen- skinned muscle fibers.42 More recently, however, these analogs have also
sitive determination of 3´,5´-cyclic AMP (cAMP) levels in cell lysates, been employed to study the effects of nucleotides on signal transduction
providing the highest sensitivity of any commercially available cAMP and to screen for compounds that may affect signal transduction, such
assay. As few as 60 femtomoles of cAMP can be detected. Furthermore, as G protein inhibitors and activators (G-Proteins and GTP Analogs for
this assay has a wide dynamic range, detecting from 0.06 to 6000 pi- Binding Studies—Note 17.1).
comoles without the need for sample dilution or manipulations such
as acetylation. This extensive dynamic range is especially important
in cell-based assays designed to measure Gs- or Gi-coupled agonist
stimulation or inhibition. Intra-assay precision for duplicate samples
is typically 5% or less.
This competitive immunoassay is formatted with maximum flex-
ibility to permit either manual assay or automated high-throughput
screening. The cAMP immunoassay is based on the highly sensitive
CSPD® alkaline phosphate substrate, a chemiluminescent 1,2-diox-
etane, with Sapphire-II™ luminescence enhancer. The ready-to-use Figure 17.3.10 BODIPY® FL forskolin (B7469).

The
TheMolecular
MolecularProbes Handbook:
Probes®
™
A Guide
Handbook: to Fluorescent
A Guide Probes
to Fluorescent and
Probes Labeling
and Technologies
Labeling Technologies
IMPORTANT NOTICE:described
The products described
manualinare
thiscovered
manual are
by covered by one or moreUse
Limited
LabelUse Label License(s).
PleasePlease
refer refer to Appendix
the Appendix
on on
IMPORTANT NOTICE : The products in this one or more Limited License(s). to the
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. 783
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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 Chapter 17 — Probes for Signal Transduction Section 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins

We prepare a variety of nucleotide analogs, including:

• Alexa Fluor® derivatives of cAMP for use as probes of type I cAMP-dependent protein ki-
nases (PKA I) and Alexa Fluor® 647 ATP (A22362)
• BODIPY® dye–labeled nucleotides for use as enzyme substrates and as long-wavelength
probes of nucleotide-binding sites
• Environment-sensitive, blue-fluorescent N-methylanthraniloyl (MANT) nucleotides
• Blue-fluorescent ethenoadenosine triphosphate (ε-ATP, E23691)
• Environment-sensitive trinitrophenyl (TNP) nucleotides
• Caged nucleotides, which are important probes for studying the kinetics and mechanism of
Figure 17.3.12 Adenosine 5’-triphosphate, BODIPY® FL nucleotide-binding proteins because they allow spatial and temporal control of the release
2’-(or-3’)-O-(N-(2-aminoethyl)urethane), trisodium salt of active nucleotide
(BODIPY® FL ATP) (A12410). • Photoaffinity nucleotides for site-selective covalent labeling
• Fluorescent ChromaTide® nucleotides and aha-dUTP nucleotides, which are primarily used
for biosynthetic incorporation into DNA or RNA (Section 8.2)

Alexa Fluor® cAMP and Alexa Fluor® ATP

Our Alexa Fluor® cAMP analogs are 8-(6-aminohexyl)amino derivatives; similar analogs
have been shown to exhibit a marked preference for binding to type I cAMP-dependent protein
1
Ex = 488 nm
kinases (PKA I). We offer the green-fluorescent Alexa Fluor® 488 cAMP (A35775, Figure 17.3.11)
and far-red–fluorescent Alexa Fluor® 647 cAMP (A35777). Alexa Fluor® 488 cAMP was loaded
Fluorescence emission

2 into cells by electroporation and then used to measure intercellular diffusion of cAMP from
regulatory to responder T cells via gap junctions.43
The Alexa Fluor® 647 conjugate of ATP (A22362) comprises the long-wavelength Alexa
Fluor® 647 fluorophore linked to the ribose of ATP by a urethane bridge. Validated applica-
tions of this probe include fluorescence resonance energy transfer (FRET) analysis (Fluorescence
3
Resonance Energy Transfer (FRET)—Note 1.2) of nucleotide assocation with Na+/K+-ATPase 44
and measurements of the catalytic activity of heavy meromyosin.45
500 550 600
Wavelength (nm) BODIPY® Ribonucleotide Di- and Triphosphates
Our selection of BODIPY® dye–modified ribonucleotides includes:
Figure 17.3.13 Fluorescence emission spectra of (1) free
BODIPY® FL dye in phosphate-buffered saline, pH 7.2;
(2) BODIPY® FL ATP (A12410); and (3) BODIPY® FL GTP • BODIPY® FL adenosine 5´-triphosphate (BODIPY® FL ATP, A12410)
(G12411). Samples were prepared with equal absorbance • BODIPY® TR adenosine 5´-triphosphate (BODIPY® TR ATP, A22352)
at the excitation wavelength (488 nm). The areas under the
curves are therefore proportional to the relative fluores- • BODIPY® TR adenosine 5´-diphosphate (BODIPY® TR ADP, A22359)
cence quantum yields, clearly showing the quenching effect • BODIPY® FL guanosine 5´-triphosphate (BODIPY® FL GTP, G12411)
caused by interaction of the BODIPY® FL fluorophore with • BODIPY® TR guanosine 5´-triphosphate (BODIPY® TR GTP, G22351)
the guanine base of GTP.
• BODIPY® FL guanosine 5´-diphosphate (BODIPY® FL GDP, G22360)

SO3 SO3
H2N O NH2

NH2 C OH
O
N N 6

NH(CH2)6NH C
N N
O 5
OCH2 O

2 (CH3CH2)3NH
O P
O O OH

Figure 17.3.14 Adenosine 5’-O-(3-thiotriphosphate), Figure 17.3.11 Alexa Fluor® 488 8-(6-aminohexyl)aminoadenosine 3’,5’-cyclicmonophosphate, bis(triethylammonium) salt
BODIPY® FL thioester, sodium salt (BODIPY® FL ATP-γ-S, (Alexa Fluor® 488 cAMP) (A35775).
thioester) (A22184).

The
The MolecularProbes®
Molecular Probes Handbook:
Handbook: A
A Guide
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™
to Fluorescent Probesand
Fluorescent Probes andLabeling
LabelingTechnologies
Technologies
IMPORTANT NOTICE: The products described in this manual aremanual
coveredare
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by Limited Use Label License(s). Please refer to thePlease
Appendix on to
784 IMPORTANT NOTICE : The products described in this covered one or more Limited Use Label License(s).
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Chapter 17 — Probes for Signal Transduction Section 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins

These mixed-isomer analogs comprise a BODIPY® fluorophore at-

tached to the 2´ or 3´ position of the ribose ring via an aminoethylcar- Electron-transfer quenching
bamoyl linker (Figure 17.3.12). Interactions between the fluorophore and e<
the purine base are evident from the spectroscopic properties of these
nucleotide analogs. The fluorescence quantum yield of BODIPY® FL O

GTP and BODIPY® FL ATP is significantly quenched in solution (Figure H C

3 HN N
17.3.13) and increases upon binding to at least some GTP-binding pro- H N N N
teins.46,47 Similar nucleotide analogs incorporating fluorophores such N
B
N O O O O 2
F F CH NH C CH S P O P O P OCH
as fluorescein, tetramethylrhodamine and Cy®3 dye have been primar- H C
3 2 2
< < < 2 O
O O O
ily used for biophysical studies of nucleotide-binding proteins.48 The
BODIPY® dye–labeled nucleotides may be particularly useful for fluo- OH OH
rescence polarization–based assays of ATP- or GTP-binding proteins. BODIPY® FL GTP-a-S
Weak fluorescence
Fhit
Nonhydrolyzable BODIPY® ATP and GTP Analogs
Among the most useful fluorescent nucleotides for protein-binding
studies are those that stoichiometrically bind to ATP- or GTP-binding H C
3

sites but are not metabolized. We offer the following nonhydrolyzable

N N
BODIPY® nucleotides: F
B
F
O O O
BODIPY® FL thiodiphosphate
H C CH NH C CH S P O P OH
3 2 2
<
O
< Strong fluorescence
O
• BODIPY® FL AMPPNP 49 (B22356)
+
• BODIPY® FL ATP-γ-S (A22184, Figure 17.3.14)
• BODIPY® FL GTP-γ-S46 (G22183) O

• BODIPY® 515/530 GTP-γ-S (G35779) HN N

• BODIPY® TR GTP-γ-S (G35780)
H N N N
• BODIPY® FL GTP-γ-NH amide (G35778) O 2
HO P OCH
GMP
< 2 O
O
The fluorescence of the BODIPY® GTP-γ-S thioesters is quenched
~90% relative to that of the free dye but is recovered upon protein bind-
OH OH
ing to G-proteins.46 The green-fluorescent BODIPY® FL GTP-γ-S has
been used to detect GTP-binding proteins separated by capillary elec-
Figure 17.3.15 Principle of fluorescence-based detection of the diadenosine triphosphate
trophoresis.50 As compared with BODIPY® FL GTP-γ-S thioester, the
hydrolase activity of Fhit using BODIPY® FL GTP-γ-S thioester (G22183) as a substrate analog.
green-fluorescent BODIPY® 515/530 GTP-γ-S thioester has a greater
fluorescence increase upon protein binding. The BODIPY® TR GTP-
γ-S thioester is a red-fluorescent analog with spectral properties similar
to the Texas Red® dye.
Although BODIPY® FL GTP-γ-NH amide exhibits less fluorescence
enhancement upon protein binding, it is reportedly the best of the three
green-fluorescent GTP-γ analogs for directly monitoring nucleotide ex-
change.51 The different linker lengths of the green-fluorescent GTP-γ
analogs (six-carbon for BODIPY® FL GTP-γ-NH amide, four-carbon for
BODIPY® FL GTP-γ-S and one-carbon for BODIPY® 515/530 GTP-γ-S)
may be useful for understanding protein active-site geometries.
In addition to their potential use for binding studies, BODIPY® FL
ATP-γ-S and BODIPY® FL GTP-γ-S thioesters are important substrates
for Fhit (Figure 17.3.15), a member of the histidine triad superfamily
of nucleotide-binding proteins that bind and cleave diadenosine poly-
phosphates.52–54 Fhit, one of the most frequently inactivated proteins
in lung cancer, functions as a tumor suppressor by inducing apopto-
sis.53,55,56 These BODIPY® nucleotides should be especially useful for
screening potential Fhit inhibitors and activators. Figure 17.3.16 2’-(or-3’)-O-(N-methylanthraniloyl)adenosine 5’-triphosphate, trisodium salt
(MANT-ATP) (M12417).

N-Methylanthraniloyl (MANT) Nucleotides

The blue-fluorescent MANT nucleotide analogs of ATP (Figure
17.3.16), AMPPNP, GTP, GMPPNP, ADP and GDP are modified on the
ribose moiety, making these probes particularly useful for studying
nucleotide-binding proteins that are sensitive to modifications of the

TheMolecular
The MolecularProbes
Probes® Handbook:
Handbook: A Guide to Fluorescent
A Guide Probes
to Fluorescent and
Probes Labeling
and Technologies
Labeling Technologies
™

IMPORTANT NOTICE: described

The products described in this
are manual
coveredare
bycovered
one or by oneLimited
or more Use
Limited UseLicense(s).
Label License(s). Please
referrefer to the Appendix
onon
IMPORTANT NOTICE : The products in this manual more Label Please to the Appendix
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. 785
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 Chapter 17 — Probes for Signal Transduction Section 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins

purine base.57,58 The compact nature of the MANT fluorophore and its attachment position on
the ribose ring results in nucleotide analogs that induce minimal perturbation of nucleotide–
protein interactions, as confirmed by X-ray crystal structures of MANT nucleotides bound to
myosin 59 and H–ras p21.60 Furthermore, because MANT fluorescence is sensitive to the envi-
ronment of the fluorophore, nucleotide–protein interactions may be directly detectable. These
properties (Table 17.2) make MANT nucleotides valuable probes of the structure and enzymatic
activity of nucleotide-binding proteins.48
Applications for MANT-ATP (M12417), MANT-ADP (M12416) and MANT-AMPPNP
(M22354) include analysis of:

Figure 17.3.17 1,N 6-ethenoadenosine 5’-triphosphate

• ATPase kinetics of kinesin 61–63 and other microtubule motor-proteins 64,65 using stopped-
(ε-ATP) (E23691). flow fluorescence measurements
• Conformation of the myosin subfragment-1 nucleotide-binding site, as indicated by fluores-
cence quencher accessibility 66,67
• Interaction of P-glycoprotein ATP-binding sites with drug efflux–modulating steroids 68
• Myosin ATPase activity in rabbit skeletal muscle 69
• Structural characteristics of the nucleotide-binding site of Escherichia coli DnaB helicase 70,71

Applications for MANT-GTP (M12415), MANT-GDP (M12414) and MANT-GMPPNP

(M22353) include analysis of:

• Activation of protein kinases by Rho subfamily GTP-binding proteins 72

• Conformational changes during activation of heterotrimeric G-proteins 73
• Effects of nucleotide structural modifications on binding to H–ras p21 74
• Nucleotide hydrolysis and dissociation kinetics of H–ras p21 and other low molecular weight
GTP-binding proteins 57,75–78
• GTP-binding proteins Rab5 and Rab7,79 Raf-1,80 Rho 81,82 and Rac,83,84 as well as Ras-related
proteins 57,75
Figure 17.3.18 2’-(or-3’)-O-(trinitrophenyl)adenosine 5’-tri-
phosphate, trisodium salt (TNP-ATP) (T7602). Ethenoadenosine Nucleotide
The ethenoadenosine nucleotides—developed in 1972 by Leonard and collaborators 85,86 —
bind like endogenous nucleotides to several proteins. The properties and applications of ethe-
noadenosine and MANT nucleotides have been comprehensively reviewed.48 The etheno ATP
analog (ε-ATP, E23691; Figure 17.3.17) can often mimic ATP in both binding and function. This
probe has been used to replace ATP in actin polymerization reactions 87 and is frequently in-
corporated in place of the tightly bound actin nucleotide.88,89 It also supports contraction of
actomyosin, facilitates the measurement of nucleotide-exchange kinetics in actin 90 and serves
as a substrate for myosin, which converts it to ε-ADP.91 Sensitized luminescence of Tb3+ (T1247,
Section 14.3) coordinated to ε-ATP is a sensitive probe of binding to the catalytic site of protein
disulfide isomerase.92

Trinitrophenyl (TNP) Nucleotides

Unlike the etheno derivatives, the free trinitrophenyl (TNP) nucleotides are essentially non-
fluorescent in water. The TNP nucleotides undergo an equilibrium transition to a semiquinoid
structure that has relatively long-wavelength spectral properties; 93–95 this form is only fluores-
cent when bound to the nucleotide-binding site of some proteins. The TNP derivative of ATP
frequently exhibits a spectral shift and fluorescence enhancement upon protein binding and
actually binds with higher affinity than ATP to some proteins. The broad, long-wavelength ab-
sorption of TNP nucleotides makes them useful for FRET studies 96–98 (Fluorescence Resonance

Table 17.2 Spectroscopic properties of MANT-nucleotides in aqueous solution (pH 8).

Parameter Value Notes
Absorption maximum 356 nm Stronger absorption at shorter wavelengths (λmax = 255 nm)
Molar extinction coefficient (ECmax) 5800 cm–1M–1 23,000 cm–1M–1 at 255 nm
Fluorescence emission maximum 448 nm Shifts 10–20 nm shorter in nonpolar solvents and upon binding to most proteins
Fluorescence quantum yield 0.22 Increases in nonpolar solvents and upon binding to most proteins

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Chapter 17 — Probes for Signal Transduction Section 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins

Energy Transfer (FRET)—Note 1.2). The TNP derivatives of ATP (TNP- sometimes difficult to properly abstract papers that describe experiments
ATP, T7602; Figure 17.3.18), ADP (TNP-ADP, T7601) and AMP (TNP- with caged ATP because they could be referring to either NPE-caged ATP
AMP, T7624) have been used as structural probes for a wide variety (A1048), DMNPE-caged ATP (A1049) or earlier caged versions of this
of nucleotide-binding proteins.99–101 We have found that chromato- nucleotide, most researchers have used NPE-caged ATP.
graphically purified TNP nucleotides are unstable during lyophiliza- Because the caged nucleotides may be added to an experimental
tion. Consequently, these derivatives are sold in aqueous solution and system at relatively high concentrations, use of the enzyme apyrase was
should be frozen immediately upon arrival. recommended by Sleep and Burton 106 to eliminate any traces of ATP
that may be present in the caged ATP probes.107–110 Once the caged ATP
Caged Nucleotides solutions have been preincubated with apyrase, the enzyme can be re-
Caged nucleotides are nucleotide analogs in which the terminal moved by centrifugal filtration.107,109
phosphate is esterified with a blocking group, rendering the molecule These caged nucleotides are generally cell impermeant and must
biologically inactive. Photolytic removal of the caging group by UV il- be microinjected into cells or loaded by other techniques (Table
lumination results in a pulse of the nucleotide—often on a microsecond 14.1). Permeabilization of cells with staphylococcal α-toxin or the
to millisecond time scale—at the site of illumination. Because photoly- saponin ester β-escin is reported to make the membrane of smooth
sis ("uncaging") can be temporally controlled and confined to the area muscle cells permeable to low molecular weight (<1000 daltons) mol-
of illumination, the popularity of this technique is growing. We are ecules, while retaining high molecular weight compounds.111 α-Toxin
supporting this development by synthesizing a variety of caged nucleo- permeabilization has permitted the introduction of caged nucleotides,
tides, neurotransmitters and Ca 2+ chelators. Our current selection of including caged ATP (A1048) and caged GTP-γ-S, as well as of caged
caged nucleotides includes: inositol 1,4,5-triphosphate (NPE-caged Ins 1,4,5; I23580; Section 17.2)
into smooth muscle cells.112 Caged inositol 1,4,5-triphosphate has also
• NPE-caged ATP (A1048) been successfully loaded in ECV304 cells using electroporation.113
• DMNPE-caged ATP (A1049)
• NPE-caged ADP (A7056) BzBzATP
• DMNB-caged c-AMP (D1037) Functional ion channels can be assembled from both homomeric
and heteromeric combinations of the seven P2X receptor subunits so
Section 5.3 discusses our selection of caged probes and the proper- far identified (P2X1–7). Due to the lack of specific agonists or antagonists
ties of the different caging groups that we use (Table 5.2). for P2X receptors, it is difficult to determine which receptor subtypes
Researchers investigating the cytoskeleton have benefited greatly mediate particular cellular responses. We offer one of the most potent
from advances in caging technology, primarily originating from the work and widely used P2X receptor agonists, BzBzATP 114,115 (2´-(or 3´-)O-(4-
of Trentham, Kaplan and their colleagues.102 NPE-caged ADP (A7056) benzoylbenzoyl)adenosine 5´-triphosphate, B22358). BzBzATP has
is a useful probe for studying the effect of photolytic release of ADP in more general applications for site-directed irreversible modification of
muscle fibers 103,104 and isolated sarcoplasmic reticulum.105 Although it is nucleotide-binding proteins via photoaffinity labeling.116,117

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1. Biochim Biophys Acta (2008) 1784:94; 2. J Am Chem Soc (2009) 131:13286; 3. Free 742:496; 59. J Mol Biol (1997) 274:394; 60. J Mol Biol (1995) 253:132; 61. Biochemistry
Radic Biol Med (2009) 47:983; 4. J Biomol Screen (1999) 4:67; 5. J Med Chem (2010) (1998) 37:792; 62. J Biol Chem (1997) 272:717; 63. Biochemistry (1995) 34:13233;
53:1898; 6. Am J Physiol (1989) 256:C886; 7. J Neurochem (1986) 47:1405; 8. Nature 64. Biochemistry (1996) 35:2365; 65. Biochemistry (1995) 34:13259; 66. Biophys J (1995)
(1986) 321:698; 9. J Neurosci (1985) 5:2672; 10. Eur J Pharmacol (1991) 207:17; 68:142S; 67. Biochemistry (1990) 29:3309; 68. Biochemistry (1997) 36:15208; 69. J Gen
11. Antimicrob Agents Chemother (2009) 53:3501; 12. J Pharm Pharmacol (2001) 53:583; Physiol (1995) 106:957; 70. Biophys J (1996) 71:2075; 71. J Cell Biol (1997) 139:63;
13. Med Res Rev (1995) 15:111; 14. Photochem Photobiol (2006) 82:720; 15. Anticancer 72. Biochemistry (1997) 36:1173; 73. J Biol Chem (1994) 269:13771; 74. Biochemistry
Res (1998) 18:4651; 16. Photochem Photobiol (1998) 68:593; 17. Proc Natl Acad Sci (1995) 34:593; 75. Biochemistry (1997) 36:4535; 76. Biochemistry (1995) 34:12543;
U S A (1989) 86:5963; 18. Pharmacol Ther (1994) 63:1; 19. J Biol Chem (2002) 277:37718; 77. Biochemistry (1995) 34:639; 78. Biochemistry (1993) 32:7451; 79. J Biol Chem (1996)
20. Photochem Photobiol (1994) 59:529; 21. J Biol Chem (2008) 283:3401; 22. Nat Med 271:20470; 80. J Biol Chem (2000) 275:22172; 81. Biochemistry (1999) 38:985; 82. J Biol
(2006) 12:549; 23. Nature (2005) 437:911; 24. Nat Cell Biol (2005) 7:78; 25. Comb Chem Chem (1996) 271:10004; 83. J Biol Chem (1997) 272:18834; 84. J Biol Chem (1996)
High Throughput Screen (2003) 6:341; 26. Proteomics (2003) 3:1244; 27. PLoS Biol (2009) 271:19794; 85. Biochem Biophys Res Commun (1972) 46:597; 86. Science (1972) 175:646;
7:e1000172; 28. J Pharmacol Exp Ther (2008) 325:27; 29. J Biol Chem (2005) 280:4048; 87. Biochemistry (1988) 27:3812; 88. J Biol Chem (2008) 283:19379; 89. J Biol Chem
30. J Med Chem (2008) 51:1831; 31. Mol Pharmacol (2008) 73:1371; 32. J Biomol (1993) 268:8683; 90. J Cell Biol (1988) 106:1553; 91. J Biol Chem (1984) 259:11920; 92. Am
Screen (2000) 5:239; 33. J Biochem (2006) 139:543; 34. Mol Endocrinol (2007) 21:700; J Physiol Lung Cell Mol Physiol (2000) 278:L1091; 93. Eur J Biochem (2003) 270:3479;
35. Kidney Int (2007) 71:738; 36. J Immunol (2005) 174:1073; 37. J Clin Invest (2003) 94. Biochim Biophys Acta (1973) 320:635; 95. Biochim Biophys Acta (1976) 453:293;
112:398; 38. J Biol Chem (2004) 279:19790; 39. Nature (2004) 429:188; 40. Proc Natl 96. J Muscle Res Cell Motil (1992) 13:132; 97. Biochemistry (1992) 31:3930; 98. Biophys
Acad Sci U S A (2004) 101:1508; 41. Nucleic Acids Res (2003) 31:e130; 42. J Cell Sci J (1992) 61:553; 99. Biochemistry (2006) 45:7237; 100. J Biol Chem (2006) 281:27471;
(2001) 114:459; 43. J Exp Med (2007) 204:1303; 44. Biochim Biophys Acta (2009) 101. Br J Pharmacol (2003) 140:202; 102. Nat Methods (2007) 4:619; 103. Biophys J (1995)
1794:1549; 45. Biochemistry (2007) 46:7233; 46. J Biol Chem (2001) 276:29275; 47. Anal 68:78S-80S; 104. J Mol Biol (1992) 223:185; 105. Ann N Y Acad Sci (1982) 402:478;
Biochem (2001) 291:109; 48. Methods Enzymol (1997) 278:363; 49. J Biol Chem (2004) 106. Biophys J (1994) 67:2436; 107. Biophys J (1994) 67:1933; 108. J Biol Chem (1995)
279:28402; 50. Anal Chem (2003) 75:4297; 51. Proc Natl Acad Sci U S A (2004) 101:2800; 270:23966; 109. Biophys J (1994) 66:1115; 110. J Biolumin Chemilumin (1994) 9:29;
52. Proc Natl Acad Sci U S A (2003) 100:1592; 53. Curr Biol (2000) 10:907; 54. J Biol 111. Methods Cell Biol (1989) 31:63; 112. Annu Rev Physiol (1990) 52:857; 113. J Neurosci
Chem (2000) 275:4555; 55. Am J Pathol (2000) 156:419; 56. J Natl Cancer Inst (2000) Methods (2004) 132:81; 114. J Physiol (1999) 519 Pt 3:723; 115. Mol Pharmacol (1999)
92:338; 57. Proc Natl Acad Sci U S A (1990) 87:3562; 58. Biochim Biophys Acta (1983) 56:1171; 116. J Neurochem (1993) 61:1657; 117. Biochemistry (1989) 28:3989.

The
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MolecularProbes
Probes®Handbook:
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A Guide Fluorescent Probes
Fluorescent Probesand
andLabeling
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IMPORTANT NOTICE: described
The productsin described in this
aremanual
coveredarebycovered
one orby one Limited
or more Limited UseLicense(s).
Label License(s). Please refer to the Appendix
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page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. 787
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 Chapter 17 — Probes for Signal Transduction Section 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins

DATA TABLE 17.3 PROBES FOR PROTEIN KINASES, PROTEIN PHOSPHATASES AND NUCLEOTIDE-BINDING PROTEINS
Cat. No. MW Storage Soluble Abs EC Em Solvent Notes
A1048 700.30 FF,D,LL H2O 259 18,000 none MeOH 1, 2, 3
A1049 760.35 FF,D,LL H2O 351 4400 none H2O 1, 2
A7056 614.44 FF,D,LL H2O 259 15,000 none MeOH 1, 2, 3
A12410 933.30 FF,L H2O 505 54,000 514 H2O 4, 5
A12412 1117.18 FF,L H2O 323 4200 461 pH 7 4, 5, 6
A22184 878.28 FF,L H2O 504 68,000 514 pH 7 4, 5
A22352 1065.43 FF,L H2O 591 55,000 620 pH 7 4, 5
A22359 963.47 FF,L H2O 592 57,000 621 pH 7 4, 5
A22362 ~2050 FF,L H2O 648 246,000 667 pH 7 4, 5
A35775 1162.23 FF,L H2O 493 71,000 517 pH 7 4, 5
A35777 ~1700 FF,L H2O 649 246,000 666 pH 7 4, 5
B7469 784.70 F,D,L DMSO 504 79,000 511 MeOH
B22356 932.31 FF,L H2O 504 68,000 514 H2O 4, 5
B22358 1018.97 FF,L H2O 260 27,000 none pH 7
D1037 524.38 F,D,LL DMSO 338 6100 none MeOH 1, 2
E23691 619.13 FF H2O 265 5000 411 pH 7 5
G12411 949.30 FF,L H2O 504 68,000 511 H2O 4, 5, 7
G22183 894.28 FF,L H2O 504 68,000 510 pH 7 4, 5, 7
G22351 1081.43 FF,L H2O 591 56,000 620 pH 7 4, 5, 7
G22360 1005.75 FF,L H2O 504 68,000 508 pH 7 4, 5, 7
G35778 905.29 FF,L H2O 505 68,000 512 pH 7 4, 5, 7
G35779 865.28 FF,L H2O 511 520 pH 7 4, 5, 7
G35780 1153.60 FF,L H2O 591 621 pH 7 4, 5, 7
H7476 504.45 F,D,L DMSO, DMF 591 37,000 594 EtOH
M12414 620.32 FF,L H2O 356 5700 447 pH 8 4, 5, 8
M12415 722.28 FF,L H2O 356 5700 448 pH 7 4, 5, 8
M12416 604.32 FF,L H2O 356 5800 448 pH 7 4, 5, 8
M12417 706.28 FF,L H2O 356 5800 447 pH 7 4, 5, 8
M22353 721.29 FF,L H2O 357 5700 447 pH 8 4, 5, 8
M22354 705.29 FF,L H2O 357 5800 447 pH 8 4, 5, 9
T7601 682.26 FF,L H2O 408 26,000 none pH 8 4, 5, 9
T7602 784.22 FF,L H2O 408 26,000 none pH 8 4, 5, 9
T7624 579.29 F,L H2O 408 26,000 none pH 8 4, 5, 9
For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.
Notes
1. Caged nucleotide esters are free of contaminating free nucleotides when initially prepared. However, some decomposition may occur during storage.
2. All photoactivatable probes are sensitive to light. They should be protected from illumination except when photolysis is intended.
3. This compound has weaker visible absorption at >300 nm but no discernible absorption peaks in this region.
4. The molecular weight (MW) of this product is approximate because the degree of hydration and/or salt form has not been conclusively established.
5. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."
6. QY = 0.63 in 50 mM Tris, pH 8.0. Fluorescence shifts to longer wavelengths (Em ~475 nm) on enzymatic cleavage of the α–β phosphoryl bond. (Biochem Biophys Res Commun (1978) 81:35, J Biol
Chem (1979) 254:12069)
7. Fluorescence of BODIPY® dye–labeled guanosine derivatives is generally weak due to base-specific intramolecular quenching. (Anal Biochem (2001) 291:109)
8. Fluorescence quantum yields of MANT nucleotides are environment-dependent. In H2O, QY is ~0.2. (Biochim Biophys Acta (1983) 742:496)
9. Trinitrophenyl nucleotides are in fact very weakly fluorescent in water (Em ~560 nm). Fluorescence is blue-shifted and more intense in organic solvents (DMSO, EtOH) and when bound to pro-
teins (Em ~540 nm). Absorption spectrum also has a second, less intense peak at about 470 nm. (Biochim Biophys Acta (1982) 719:509)

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Molecular Probes Handbook:
Handbook: AAGuide
Guideto
toFluorescent
™
Fluorescent Probes and Labeling
Probes and LabelingTechnologies
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IMPORTANT NOTICE: The products described in this manual
in thisaremanual
coveredare
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one or more Limited Use Label License(s). Please refer to thePlease
Appendix onto
788 IMPORTANT NOTICE : The products described by one or more Limited Use Label License(s).
the Appendix onrefer
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page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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Chapter 17 — Probes for Signal Transduction Section 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins

PRODUCT LIST 17.3 PROBES FOR PROTEIN KINASES, PROTEIN PHOSPHATASES AND NUCLEOTIDE-BINDING PROTEINS
Cat. No. Product Quantity
A22359 adenosine 5’-diphosphate, BODIPY® TR 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), disodium salt (BODIPY® TR ADP) *5 mM in buffer* 100 µL
A7056 adenosine 5’-diphosphate, P2-(1-(2-nitrophenyl)ethyl) ester, monopotassium salt (NPE-caged ADP) 5 mg
A22184 adenosine 5’-O-(3-thiotriphosphate), BODIPY® FL thioester, sodium salt (BODIPY® FL ATP-γ-S, thioester) *5 mM in buffer* 50 µL
A22362 adenosine 5’-triphosphate, Alexa Fluor® 647 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), hexa(triethylammonium) salt (Alexa Fluor® 647 ATP) *5 mM in buffer* 100 µL
A12410 adenosine 5’-triphosphate, BODIPY® FL 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), trisodium salt (BODIPY® FL ATP) *5 mM in buffer* 100 µL
A22352 adenosine 5’-triphosphate, BODIPY® TR 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), trisodium salt (BODIPY® TR ATP) *5 mM in buffer* 100 µL
A1048 adenosine 5’-triphosphate, P3-(1-(2-nitrophenyl)ethyl) ester, disodium salt (NPE-caged ATP) 5 mg
A1049 adenosine 5’-triphosphate, P3-(1-(4,5-dimethoxy-2-nitrophenyl)ethyl) ester, disodium salt (DMNPE-caged ATP) 5 mg
A12412 adenosine 5’-triphosphate, P3-(5-sulfo-1-naphthylamide), tetra(triethylammonium) salt (ATP γ-AmNS) *5 mM in buffer* 400 µL
A35775 Alexa Fluor® 488 8-(6-aminohexyl)aminoadenosine 3’,5’-cyclicmonophosphate, bis(triethylammonium) salt (Alexa Fluor® 488 cAMP) *5 mM in buffer* 100 µL
A35777 Alexa Fluor® 647 8-(6-aminohexyl)aminoadenosine 3’,5’-cyclicmonophosphate, tetra(triethylammonium) salt (Alexa Fluor® 647 cAMP) *5 mM in buffer* 100 µL
A35725 Antibody Beacon™ Tyrosine Kinase Assay Kit *400 assays* 1 kit
A6442 anti-synapsin I (bovine), rabbit IgG fraction *affinity purified* 10 µg
B22358 2’-(or-3’)-O-(4-benzoylbenzoyl)adenosine 5’-triphosphate, tris(triethylammonium) salt (BzBzATP) *5 mM in buffer* 2 mL
B7469 BODIPY® FL forskolin 100 µg
B22356 2’-(or-3’)-O-(BODIPY® FL)-β:γ-imidoadenosine 5’-triphosphate, trisodium salt (BODIPY® FL AMPPNP) *5 mM in buffer* 50 µL
C10558 cAMP Chemiluminescent Immunoassay Kit *10-plate size* 1 kit
C10557 cAMP Chemiluminescent Immunoassay Kit *2-plate size* 1 kit
D1037 4,5-dimethoxy-2-nitrobenzyl adenosine 3’,5’-cyclicmonophosphate (DMNB-caged cAMP) 5 mg
E23691 1,N6-ethenoadenosine 5’-triphosphate (ε-ATP) *5 mM in buffer* 2 mL
G22360 guanosine 5’-diphosphate, BODIPY® FL 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), bis(triethylammonium) salt (BODIPY® FL GDP) *5 mM in buffer* 100 µL
G35778 guanosine 5’-O-(3-iminotriphosphate), BODIPY® FL ethylamide, sodium salt (BODIPY® FL GTP-γ-NH, amide) *1 mM in buffer* 100 µL
G35779 guanosine 5’-O-(3-thiotriphosphate), BODIPY® 515/530 thioester, sodium salt (BODIPY® 515/530 GTP-γ-S, thioester) *1 mM in buffer* 100 µL
G22183 guanosine 5’-O-(3-thiotriphosphate), BODIPY® FL thioester, sodium salt (BODIPY® FL GTP-γ-S, thioester) *5 mM in buffer* 50 µL
G35780 guanosine 5’-O-(3-thiotriphosphate), BODIPY® TR thioester, sodium salt (BODIPY® TR GTP-γ-S, thioester) *1 mM in buffer* 100 µL
G12411 guanosine 5’-triphosphate, BODIPY® FL 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), trisodium salt (BODIPY® FL GTP) *5 mM in water* 100 µL
G22351 guanosine 5’-triphosphate, BODIPY® TR 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), trisodium salt (BODIPY® TR GTP) *5 mM in water* 100 µL
H7476 hypericin 1 mg
M12416 2’-(or-3’)-O-(N-methylanthraniloyl)adenosine 5’-diphosphate, disodium salt (MANT-ADP) *5 mM in buffer* 400 µL
M12417 2’-(or-3’)-O-(N-methylanthraniloyl)adenosine 5’-triphosphate, trisodium salt (MANT-ATP) *5 mM in buffer* 400 µL
M12414 2’-(or-3’)-O-(N-methylanthraniloyl)guanosine 5’-diphosphate, disodium salt (MANT-GDP) *5 mM in buffer* 400 µL
M12415 2’-(or-3’)-O-(N-methylanthraniloyl)guanosine 5’-triphosphate, trisodium salt (MANT-GTP) *5 mM in buffer* 400 µL
M22354 2’-(or-3’)-O-(N-methylanthraniloyl)-β:γ-imidoadenosine 5’-triphosphate, trisodium salt (MANT-AMPPNP) *5 mM in buffer* 50 µL
M22353 2’-(or-3’)-O-(N-methylanthraniloyl)-β:γ-imidoguanosine 5’-triphosphate, trisodium salt (MANT-GMPPNP) *5 mM in buffer* 50 µL
P13235 polymyxin B, BODIPY® FL conjugate, trifluoroacetic acid salt *mixed species* 100 µg
P13238 polymyxin B, dansyl conjugate, trifluoroacetic acid salt *mixed species* 100 µg
P13236 polymyxin B, Oregon Green® 514 conjugate, trifluoroacetic acid salt *mixed species* 100 µg
P33706 Pro-Q® Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit 1 kit
R33700 RediPlate™ 96 EnzChek® Serine/Threonine Phosphatase Assay Kit *one 96-well microplate* 1 kit
R22067 RediPlate™ 96 EnzChek® Tyrosine Phosphatase Assay Kit *one 96-well microplate* 1 kit
T7601 2’-(or-3’)-O-(trinitrophenyl)adenosine 5’-diphosphate, disodium salt (TNP-ADP) *5 mg/mL in water* 2 mL
T7624 2’-(or-3’)-O-(trinitrophenyl)adenosine 5’-monophosphate, sodium salt (TNP-AMP) *5 mg/mL in buffer* 2 mL
T7602 2’-(or-3’)-O-(trinitrophenyl)adenosine 5’-triphosphate, trisodium salt (TNP-ATP) *5 mg/mL in buffer* 2 mL

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TheMolecular
MolecularProbes
Probes®
™
Handbook: A Guide
Handbook: to Fluorescent
A Guide Probes
to Fluorescent and
Probes Labeling
and Technologies
Labeling Technologies
IMPORTANT NOTICE:described
The products described in are
this covered
manual are
bycovered by oneLimited
or moreUse
Limited UseLicense(s).
Label License(s). Please
referrefer to the Appendix
onon
IMPORTANT NOTICE : The products in this manual one or more Label Please to the Appendix
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. 789
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 Chapter 17 — Probes for Signal Transduction Section 17.4 Probes for Lipid Metabolism and Signaling

17.4 Probes for Lipid Metabolism and Signaling

PLA Lipids and lipid metabolites are abundant in cells and have both a structural function and
1
O a role in cell regulation. Phospholipases, in particular, play an important part in cellular signal-
1 ing processes via the generation of second messengers such as diacylglycerols, arachidonate and
R C OCH PLD
2
2 inositol 1,4,5-triphosphate 1–4 (Ins 1,4,5-P3, I3716; Section 17.2). In addition, phospholipase A 2
R C OCH O activation is a key step in inflammation processes, and phospholipase A 2 plays major roles in
3
O CH O P O R
2 _
bacterial virulence and in the pathogenesis of acute respiratory distress syndrome 5–7 (ARDS),
PLA
2 O making this class of enzymes important therapeutic targets.8,9
PLC Phospholipases are classified according to the cleavage site on the phospholipid substrate
(Figure 17.4.1). There are at least three types of fluorescence-based phospholipase detection
Figure 17.4.1 Cleavage specificities of phospholipas-
es. R1 and R2 are typically saturated or unsaturated aliphatic methods: 10
groups. The polar head group R3 can be choline, ethanol- • Continuous methods, which permit direct fluorometric monitoring of enzymatic activity
amine, glycerol, inositol, inositol phosphate, serine or using self-quenching or excimer-forming probes
other alcohols.
• Methods that continuously detect nonfluorescent product formation from natural phospho-
lipids, such as detection of fatty acids with our ADIFAB reagent or enzyme-coupled detec-
tion of choline with our Amplex® Red Phospholipase Assay Kits
• Discontinuous methods, which require resolution of fluorescent substrates and products by
TLC, HPLC or other separation techniques

Table 17.3 summarizes Molecular Probes® products for fluorescence-based phospholipase

assays. Other applications for our wide range of fluorescent phospholipids are described in
Chapter 13.

Table 17.3 Fluorescence-based phospholipase assays.

Phospholipase * Probes Assay Principle Detection Method
A1 A10070, E10219, E10221 Intramolecular self-quenching Fluorescence increase at ~530 nm
A 1, A 2 B7701 Intramolecular self-quenching Fluorescence increase at ~515 nm 1
A 1, A 2 B3781, B3782 Intramolecular excimer formation Emission ratio 380/470 nm 2–4
A 1, A 2 A3880 Free fatty acid sensor Emission ratio 432/v505 nm 5–7
A2 A10072, E10217, E10218 Intramolecular fluorescence resonance energy transfer Fluorescence increase at ~515 nm or increase in emission ratio at
(FRET) 515/575 nm
A2 D23739 Intramolecular self-quenching Fluorescence increase at ~515 nm 8
A2 N3786, N3787 Intermolecular self-quenching Fluorescence increase at ~530 nm 9,10
A2 H361, H3809 Intermolecular excimer formation Emission ratio 380/470 nm 11–13
A2 D3803 Release of a fluorescent fatty acid TLC or fluorescence image scanner 14
A2, C, D D3771 Formation of a fluorescent O-alkylglycerol derivative TLC or HPLC 15–17
A2, C, D H361 Quenching by a disulfide-polymerized lipid matrix Fluorescence increase at ~380 nm 18,19
C A12218 Peroxidase-linked detection of phosphocholine Conversion of the nonfluorescent Amplex® Red reagent to
fluorescent resorufin 20–22
C E10215, E10216 Release of dye-labeled diacylglycerol Fluorescence increase at 516 nm, with potential interference from
phospholipase A2 and phospholipase D activity
D A12219 Peroxidase-linked detection of choline Conversion of the nonfluorescent Amplex® Red reagent to
fluorescent resorufin 20,22,23
PAP † D3805 Release of dye-labeled diacylglycerol HPLC 24
* Phospholipase specificity: A1, A2, C or D (see Section 17.4 for cleavage specificities). † PAP = phosphatidic acid phosphohydrolase.
1. J Biol Chem (1992) 267:21465; 2. Biochemistry (1993) 32:583; 3. Anal Biochem (1981) 116:553; 4. Biochim Biophys Acta (1994) 1192:132; 5. Anal Biochem (1995) 229:256; 6. Biochem J (1994)
298:23; 7. J Biol Chem (1992) 267:23495; 8. Anal Biochem (1999) 276:27; 9. Lipids (1989) 24:691; 10. Biochem Biophys Res Commun (1984) 118:894; 11. Anal Biochem (2006) 359:280; 12. Chem
Phys Lipids (1990) 53:129; 13. Anal Biochem (1989) 177:103; 14. J Biol Chem (1999) 274:19338; 15. Eukaryot Cell (2009) 8:1094; 16. Anal Biochem (1994) 218:136; 17. Biochem J (1995) 307:799;
18. Anal Biochem (1994) 221:152; 19. J Biol Chem (1995) 270:263; 20. Proc Natl Acad Sci U S A (2004) 101:9745; 21. Mol Pharmacol (2000) 57:1142; 22. Proc SPIE-Int Soc Opt Eng (2000) 3926:166;
23. J Biol Chem (2002) 277:45592; 24. Anal Biochem (2008) 374:291.

The
The MolecularProbes®
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Handbook: A
A Guide
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™
to Fluorescent Probesand
Fluorescent Probes andLabeling
LabelingTechnologies
Technologies
IMPORTANT NOTICE: The products described in this manual
in thisaremanual
coveredare
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Appendix onto
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Chapter 17 — Probes for Signal Transduction Section 17.4 Probes for Lipid Metabolism and Signaling

Phospholipase A1 and A2 Assays

ester linkage between phospholipids and fatty acids, Figure 17.4.1), and
The importance of phospholipases in cellular signaling, lipid me- the fluorescence response associated with enzymatic cleavage of the
tabolism, inflammatory responses and pathological disorders related to substrate (Table 17.3).
these processes has stimulated demand for fluorescence-based enzyme
activity monitoring methods. Several of the fluorogenic phospholipase PED-A1 Phospholipase A1 Substrate
A substrates described here are designed to provide continuous moni- PED-A1 (A10070, Figure 17.4.3) is a fluorogenic substrate designed
toring of phospholipase A activity in purified enzyme preparations, cell to provide specific, real-time monitoring of phospholipase A1 activity
lysates and live cells; applications of some of these substrates extend as in purified enzyme preparations, cell lysates and live cells.14,15 PED-A1
far as in vivo small animal imaging.11–13 The phospholipase A substrates is comprised of a dinitrophenyl quencher–modified glycerophospho-
are generally dye-labeled phospholipids of two types—glycerophospho- ethanolamine head group and a green-fluorescent BODIPY® FL dye–
cholines with BODIPY® dye–labeled sn-1 or sn-2 (or both) acyl or alkyl labeled acyl chain at the sn-1 position. Upon cleavage by phospholipase
chains and glycerophosphoethanoloamines with BODIPY® dye–labeled A1, PED-A1 exhibits an increase in green fluorescence (measured at
acyl chains and dinitrophenyl quencher–modified head groups (Figure excitation/emission = 488/530 nm). Phospholipase A1 specificity is im-
17.4.2). These structural variations determine specificity for phospholi- parted by the placement of the BODIPY® FL acyl chain in the sn-1 posi-
pase A1 (which hydrolyzes the sn-1 ester linkage between phospholipids tion and by incorporation of an acyl group with an enzyme-resistant
and fatty acids) versus phospholipase A 2 (which hydrolyzes the sn-2 (noncleavable) ether linkage in the sn-2 position.

H C
3 Quenched substrate Quenched substrate
(bis-BODIPY FL C11-PC) (PED6)
N N
B O O
H C F F (CH ) C OCH CH (CH ) C OCH
3 2 10 2 3 2 14 2 O N
2
H C F F (CH ) C OCH O H C F F (CH ) C OCH O O
3 2 10 + 3 24
B O CH O P OCH CH N(CH ) B O CH O P OCH CH NH C (CH ) NH NO
N N 2 < 2 2 33 N N 2 < 2 2 25 2
O O
Phospholipase A 2 Phospholipase A 2
H C H C
3 3

H C
3
O
CH (CH ) C OCH O N
3 2 14 2 2
N N
B O HOCH O O
H C F F (CH ) C OCH CH O P OCH CH NH C (CH ) NH NO
3 2 10 2 2 < 2 2 25 2
HOCH O O
+
CH O P OCH CH N(CH )
2 2 2 33
Fluorescent lysophospholipid O
< Nonfluorescent lysophospholipid

+ +

H C F F (CH ) C OH H C F F (CH ) C OH
3 2 10 3 24
B O B O
N N N N

H C H C
3 3

Fluorescent fatty acid (BODIPY FL C11 (D-3862)) Fluorescent fatty acid (BODIPY FL C5 (D-3834))
Figure 17.4.2 Mechanism of phospholipase activity–linked fluorescence enhancement responses of bis-BODIPY® FL C11-PC (B7701) and PED6
(D23739). Note that enzymatic cleavage of bis-BODIPY® FL C11-PC yields two fluorescent products, whereas cleavage of PED6 yields only one.

H3C

N N O
�
H3C � � (CH2)� C OCH2
O2N
CH3(CH2)� OCH O O
CH2O P OCH2CH2NH C (CH2)�NH NO2
OH

Figure 17.4.3 PED-A1 (N-((6-(2,4-DNP)amino)hexanoyl)-1-(BODIPY® FL C5)-2-hexyl-sn-glycero-3-phosphoethanolamine, A10070).

The
TheMolecular
MolecularProbes Handbook:
Probes®
™
A Guide
Handbook: to Fluorescent
A Guide Probes
to Fluorescent and
Probes Labeling
and Technologies
Labeling Technologies
IMPORTANT NOTICE:described
The products described
manualinare
thiscovered
manual are
by covered by one or moreUse
Limited
LabelUse Label License(s).
PleasePlease
refer refer to Appendix
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 Chapter 17 — Probes for Signal Transduction Section 17.4 Probes for Lipid Metabolism and Signaling

8,000
EnzChek® Phospholipase A1 Assay Kit
7,000 The EnzChek® Phospholipase A1 Assay Kit (E10219, E10221) provides a simple, fluorometric
Fluorescence intensity

6,000 method for continuous monitoring of phospholipase A1 activity based on the phospholipase
5,000 A1–specific PED-A1 substrate (A10070, described above). The EnzChek® Phospholipase A1 Assay
4,000 3,500
Kit can detect phospholipase A1 activity at 0.04 U/mL or lower (Figure 17.4.4). This microplate-
based assay is well suited for rapid and direct analysis of phospholipase A1 in purified enzyme
3,000
2,500
3,000 2,000

2.000
1,500
1,000
preparations and cell lysates using automated instrumentation, as well as for characterizing
500
0 phospholipase A1 inhibitors.
1,000 0 0.2 0.4 0.6 0.8 1 1.2
Each EnzChek® Phospholipase A1 Assay Kit (2-plate size, E10219; 10-plate size, E10221) provides:
0
0 1 2 3 4 5
PLA1 concentration (U/mL) • PED-A1 phospholipase A1 substrate
Figure 17.4.4 Detection of phospholipase A1 (PLA1) using • Phospholipase A1 (Lecitase Ultra)
the EnzChek® Phospholipase A1 Assay Kit (E10219, • Concentrated phospholipase A1 reaction buffer
E10221). PLA1 reactions were run at ambient temperature • Dioleoylphosphatidylcholine (DOPC)
with liposomes for 30 minutes according to the assay pro-
tocol provided, and fluorescence emission was measured • Dioleoylphosphatidylglycerol (DOPG)
using 460 nm excitation on a Spectra Max M5 (Molecular • Dimethylsulfoxide (DMSO)
Devices). Background fluorescence determined for the no- • Detailed assay protocols
enzyme control reaction has been subtracted.

The 2-plate assay kit provides sufficient reagents for 200 reactions in 96-well microplates at a
volume of 100 µL per well or 800 reactions using low-volume 384-well microplates at a volume of
≤25 µL per well. The 10-plate assay kit provides sufficient reagents for 1000 reactions in 96-well
1,000 microplates at a volume of 100 µL per well or 4000 reactions using low-volume 384-well micro-
900 66 min
21 min
plates at a volume of ≤25 µL per well.
800 11 min
6 min
700 1 min
0 min
Red/Green BODIPY® PC-A2 Ratiometric Phospholipase A2 Substrate
600
Red/Green BODIPY® PC-A2 (A10072, Figure 17.4.5) is a ratiometric fluorogenic substrate
Intensity

500
designed to provide selective, real-time monitoring of phospholipase A 2 activity in purified
400
300
enzyme preparations, cell lysates and live cells. Cleavage of the BODIPY® FL pentanoic acid
200 substituent at the sn-2 position results in decreased quenching by fluorescence resonance en-
100 ergy transfer (FRET) of the BODIPY® 558/568 dye attached at the sn-1 position. Thus, upon
0 cleavage by phospholipase A 2, Red/Green BODIPY® PC-A2 exhibits an increase in BODIPY® FL
500 520 540 560 580 600 620 640
490.0 650.5 fluorescence, detected from 515–545 nm (Figure 17.4.6). The FRET-sensitized BODIPY® 558/568
Emission (nm)
fluorescence signal is expected to show a reciprocal decrease; in practice, however, this longer-
Figure 17.4.6 Fluorescence emission spectra (excitation wavelength fluorescence may show a decrease or a slight increase, depending on the formulation
at 480 nm) of Red/Green BODIPY® PC-A2 phospholipase A2 of the substrate and the instrument wavelength settings. The ratiometric detection mode of this
substrate (A10072) incorporated in liposomes with addition
of bee venom phospholipase A2 at ambient temperature. substrate (emission intensity ratio at 515⁄575 nm with excitation at ~460 nm) allows measure-
ments of phospholipase A 2 activity that are essentially independent of instrumentation and assay
conditions. The dual-emission properties of this substrate also provide the capacity to localize
the lysophospholipid and fatty acid products of the phospholipase A 2 cleavage via their distinct
spectroscopic signatures in imaging experiments.

EnzChek® Phospholipase A2 Assay Kit

The EnzChek® Phospholipase A2 Assay Kit (E10217, E10218) provides a simple, fluoromet-
ric method for continuous monitoring of phospholipase A 2 activity based on the phospholipase
A2–selective Red/Green BODIPY® PC-A2 (A10072, described above). This phospholipase A 2 as-
say can be used in an intensity-based detection mode, by following the fluorescence increase at

N N O
�
� � CH2CH2 C NH(CH2)6 OCH2
S
H3C � � (CH2)� C OCH O
�
N N O CH2O P OCH2CH2N(CH3)3
O

H3 C

Figure 17.4.5 Red/Green BODIPY® PC-A2 (1-O-(6-BODIPY® 558/568-aminohexyl)-2-BODIPY® FL C5-sn-glycero-3-phosphocholine,

A10072).

TheMolecular
The MolecularProbes®
Probes Handbook:
Handbook: AAGuide
Guideto
toFluorescent
Fluorescent Probes
™
and Labeling
Probes and LabelingTechnologies
Technologies
IMPORTANT NOTICE: The products described in this manual
in thisare covered bycovered
one or more Limited Use Label License(s). Please refer to thePlease
Appendix
referonto
792 IMPORTANT NOTICE : The products described manual are by one or more Limited Use Label License(s).
the Appendix on
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Chapter 17 — Probes for Signal Transduction Section 17.4 Probes for Lipid Metabolism and Signaling

~515 nm, or in a ratiometric-based detection mode, by following the

7
changes in the emission intensity ratio at 515/575 nm with excitation
6
at ~460 nm (Figure 17.4.7). The EnzChek® Phospholipase A2 Assay Kit

Fluorescence ratio
can detect bee venom phospholipase A2 activity at 0.05 U/mL or lower 5

(Figure 17.4.7). This microplate-based assay is well suited for rapid and 4

direct analysis of phospholipase A 2 in purified enzyme preparations and

5.0
4.5
3 4.0
3.5
cell lysates using automated instrumentation, as well as for character- 2
3.0
2.5
2.0

izing phospholipase A2 inhibitors. 1.5

1.0
0.5
1
Each EnzChek® Phospholipase A 2 Assay Kit (2-plate size, E10217;
0
0 0.2 0.4 0.6 0.8 1

10-plate size, E10218) provides: 0

0 1 2 3 4 5
PLA2 concentration (U/mL)
• Red/Green BODIPY® PC-A2 phospholipase A 2 substrate
3,500
• Phospholipase A 2 from honey bee venom
• Concentrated phospholipase A 2 reaction buffer 3,000

Fluorescence intensity
• Dioleoylphosphatidylcholine (DOPC) 2,500

• Dioleoylphosphatidylglycerol (DOPG) 2,000

• Dimethylsulfoxide (DMSO) 1,500
1,600
1,400
1,200

• Detailed assay protocols 1,000

800
1,000 600
400
200

The 2-plate assay kit provides sufficient reagents for 200 reactions in 500 0
0 0.2 0.4 0.6 0.8 1

96-well microplates at a volume of 100 µL per well or 800 reactions us- 0

0 1 2 3 4 5
ing low-volume 384-well microplates at a volume of ≤25 µL per well. The PLA2 concentration (U/mL)
10-plate assay kit provides sufficient reagents for 1000 reactions in 96-
Figure 17.4.7 Detection of phospholipase A2 (PLA2) using the EnzChek® Phospholipase A2
well microplates at a volume of 100 µL per well or 4000 reactions using Assay Kit (E10217, E10218). PLA2 reactions were run at ambient temperature with liposomes
low-volume 384-well microplates at a volume of ≤25 µL per well. for 10 minutes according to the assay protocol provided, and fluorescence emission was
measured using 460 nm excitation on a Spectra Max® M5 (Molecular Devices). Background
fluorescence determined for the no-enzyme control reaction has been subtracted. Top panel
PED6 Phospholipase A2 Substrate shows ratiometric-based (515/575 nm) detection mode; bottom panel shows intensity-based
PED6 (D23739, Figure 17.4.8) is a fluorogenic substrate for phos- (515 nm channel) detection mode. Background fluorescence determined for the no-enzyme
pholipase A 2 incorporating a BODIPY® FL dye–labeled sn-2 acyl control reaction has been subtracted for each value.
chain and a dinitrophenyl quencher–labeled head group 16 (Figure
17.4.2). Cleavage of the dye-labeled acyl chain by phospholipase A 2
eliminates the intramolecular quenching effect of the dinitrophenyl
group, resulting in a corresponding fluorescence increase. Continuous O
CH3(CH2)�� C OCH2
kinetic assays show PED6 to be a good substrate for both secreted O2N
and cytosolic phospholipase A 2 and platelet-activating factor acetyl- H3C � � (CH2)� C OCH O O
�
hydrolase.16 PED6 has been used by Steven Farber and co-workers for N N O CH2O P OCH2CH2NH C (CH2)�NH NO2
in vivo analysis of intestinal lipid metabolism in zebrafish larvae as a O

basis for identifying and screening mutant phenotypes 12,13,17 (Figure H3C
(CH3CH2)3NH
17.4.9). PED6 is also useful for high-throughput screening of potential
phospholipase A 2 inhibitors or activators. Figure 17.4.8 N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-2-(4,4-difluoro-5,7-dimethyl-4-bora-
3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphoethanolamine,
triethylammonium salt (PED6) (D23739).
Other BODIPY® Dye Phospholipase A Substrates
The bis-BODIPY® phospholipase A substrate—bis-BODIPY® FL
glycerophosphocholine (bis-BODIPY® FL C11-PC, B7701)—has been
specifically designed to allow continuous monitoring of phospho-
lipase A action and to be spectrally compatible with argon-ion laser
excitation sources.18 When this probe is incorporated into cell mem-
branes, the proximity of the BODIPY® FL fluorophores on adjacent
phospholipid acyl chains causes fluorescence self-quenching (Figure
17.4.2). Separation of the fluorophores upon hydrolytic cleavage of one
of the acyl chains by either phospholipase A1 or A 2 results in increased
fluorescence. Bis-BODIPY® FL C11-PC has been developed in collabora-
tion with Elizabeth Simons, who has successfully employed it for flow Figure 17.4.9 Imaging of lipid digestion pathways in zebrafish (Danio rerio) using the fluoro-
cytometric detection of phospholipase A activity in neutrophils.19 More genic phospholipase A2 substrate PED6 (D23739). A zebrafish larva (5 days post-fertilization)
recently, bis-BODIPY® FL C11-PC has been used to detect phospholipase was incubated with PED6 for 2 hours. Localized fluorescence in the gallbladder and intestinal
lumen results from endogenous lipase activity and rapid transport of the substrate cleav-
A 2 activation induced by tumor necrosis factor (TNF) 20 and for high- age products through the intestinal and hepatobiliary systems. The image was provided by
throughput assays of endothelial lipase, a critical determinant of HDL Steven A. Farber, Thomas Jefferson University.
cholesterol levels.21,22

The
TheMolecular
MolecularProbes Handbook:
Probes®
™
A Guide
Handbook: to Fluorescent
A Guide Probes
to Fluorescent and
Probes Labeling
and Technologies
Labeling Technologies
IMPORTANT NOTICE:described
The products described
manualinare
thiscovered
manual are
by covered by one or moreUse
Limited
LabelUse Label License(s).
PleasePlease
refer refer to Appendix
the Appendix
on on
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 Chapter 17 — Probes for Signal Transduction Section 17.4 Probes for Lipid Metabolism and Signaling

Specificity for phospholipase A 2 versus phospholipase A1 can be obtained using phospho-

lipids with nonhydrolyzable, ether-linked alkyl chains in the sn-1 position. A 1-O-alkyl–substi-
tuted phospholipid containing the BODIPY® FL fluorophore (D3771, Figure 17.4.10) is a useful
substrate for a phospholipase A 2–specific chromatographic assay.23
The singly labeled BODIPY® phospholipase A 2 substrate—β-BODIPY® FL C5-HPC (D3803)—
has been used to quantitatively delineate a discontinuous increase of Ca 2+-dependent cytosolic
Figure 17.4.10 2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimeth- phospholipase A 2 (cPLA 2) activity during zebrafish embryogenesis. The analytical method devel-
yl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)undecyl)-
sn-glycero-3-phosphocholine (D3771). oped for this study uses a fluorescence image scanner to quantitatively detect the free BODIPY®
FL dye–labeled fatty acid generated by the action of cPLA 224 (Figure 17.4.11).

Bis-Pyrenyl Phospholipase A Substrates

Our bis-pyrenyl phospholipase A probes (B3781, B3782) both emit at ~470 nm, indicat-
sP k

2
cP 2
LA
LA

ing that their adjacent pyrene fluorophores (Figure 17.4.12) form excited-state dimers (Figure
an
Bl

1 2 3 17.4.13). Phospholipase A–mediated hydrolysis separates the fluorophores, which then emit as
monomers at ~380 nm.25 These substrates have proven to be effective phospholipase A2 sub-
FFA product strates in model membrane systems (Table 17.3); however, it has been reported that 1,2-bis-(1-
pyrenebutanoyl)-sn-glycero-3-phosphocholine (B3781) is highly resistant to degradation by phos-
pholipases in human skin fibroblasts.26 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine
has been used in a sensitive, continuous assay for lecithin:cholesterol acyltransferase 27,28 (LCAT).
Phospholipid
substrate
Singly Labeled Pyrenyl and NBD Phospholipase A2 Substrates
Phospholipase A 2 activity has also been measured using phospholipids labeled with a single
pyrene (H361, Figure 17.4.14; H3809, Figure 17.4.15) or NBD (N3786; N3787, Figure 17.4.16) fluo-
rophore (Table 17.3). Because only the sn-2 phospholipid acyl chain is labeled, these probes can
discriminate between phospholipase A 2 and phospholipase A1 activity. To obtain a direct fluo-
rescence response to enzymatic cleavage, sufficient phospholipid must be loaded into membranes
to cause either intermolecular self-quenching (NBD-acyl phospholipids) or excimer formation 29
Figure 17.4.11 Assay of cytoplasmic phospholipase A2
(cPLA2) using β-BODIPY® FL C5-HPC (D3803) as a sub- (pyreneacyl phospholipids). Pyrene-labeled acidic phospholipids—particularly the phosphoglyc-
strate. The substrate was incubated in enzyme-free assay erol derivative 30,31 (H3809)—are preferred as substrates by pancreatic and intestinal phospho-
buffer (lane 1), with secreted PLA2 (from Naja mossambica; lipase A 2, whereas labeled phosphocholine (H361, Figure 17.4.14) is preferred by phospholipase
lane 2) or with purified human recombinant cPLA2 (lane
3). Cleavage products were separated by thin-layer chro- A 2 from snake venom.32
matography in chloroform/methanol/acetic acid/water
([Link]) and were subsequently analyzed using a fluores- ADIFAB Indicator: A Different View of Phospholipase A Activity
cence image scanner. Both phospholipases liberated fluores-
cent BODIPY® FL dye–labeled fatty acids (FFA) by cleavage of The ADIFAB fatty acid indicator (A3880, Figure 17.4.17) functions as a fluorescent sensor for
the substrate at the sn-2 acyl bond. Figure reproduced with the free fatty acid cleavage products of phospholipases. 33–36 It does not require membrane loading
permission from J Biol Chem (1999) 274:19338.
Fluorescence emission

Figure 17.4.14 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-

2
glycero-3-phosphocholine (β-py-C10-HPC) (H361).
3

350 400 450 500 550 600

Wavelength (nm)

Figure 17.4.12 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3- Figure 17.4.13 Excimer formation by pyrene in etha-

phosphocholine (B3781). nol. Spectra are normalized to the 371.5 nm peak of the
monomer. All spectra are essentially identical below 400 nm
after normalization. Spectra are as follows: 1) 2 mM pyrene,
purged with argon to remove oxygen; 2) 2 mM pyrene, air-
equilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM
pyrene (argon-purged). The monomer-to-excimer ratio Figure 17.4.15 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-
(371.5 nm/470 nm) is dependent on both pyrene concentra- glycero-3-phosphoglycerol, ammonium salt (β-py-C10-PG)
tion and the excited-state lifetime, which is variable because (H3809).
of quenching by oxygen.

The
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Probes Handbook:
Handbook: AAGuide
GuidetotoFluorescent
Fluorescent Probes and Labeling
Probes and Labeling Technologies
Technologies
™

IMPORTANT NOTICE: The products described in this manual

in thisare covered
arebycovered
one or more Limited Use Label License(s). Please refer to the Appendix
referonto
794 IMPORTANT NOTICE : The products described manual by one or more Limited Use Label License(s). Please
the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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Chapter 17 — Probes for Signal Transduction Section 17.4 Probes for Lipid Metabolism and Signaling

and can be used to monitor hydrolysis of natural (rather than synthetic) substrates. Assaying ly-
sophospholipase activity with ADIFAB yields sensitivity comparable to radioisotopic methods. 37
Richieri and Kleinfeld have described a methodology for using the ADIFAB reagent to measure
the activity of phospholipase A 2 on cell and lipid-vesicle membranes; their assay is capable of
detecting hydrolysis rates as low as 10 –12 mole/minute.36

Phospholipase C Assays
EnzChek® Direct Phospholipase C Assay Kit Figure 17.4.16 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
The EnzChek® Direct Phospholipase C Assay Kit (E10215, E10216) provides a simple and amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phospho-
choline (NBD C12-HPC) (N3787).
robust microplate-based method for monitoring phosphatidylcholine-specific phospholipase C
(PC-PLC) activity in purified enzyme preparations. PC-PLC plays a crucial role in many cell
signaling pathways involved in apoptosis and cell survival, as well as in diseases as diverse as
cancer and HIV. This assay uses a proprietary substrate (glycerophosphoethanolamine with a
dye-labeled sn-2 acyl chain) to detect PC-PLC activity. Substrate cleavage by PC-PLC releases
dye-labeled diacylglycerol, which produces a positive fluorescence signal that can be measured
continuously using a fluorescence microplate reader. The reaction product has fluorescence ex-
citation and emission maxima of 509 nm and 516 nm, respectively.
The EnzChek® Direct Phospholipase C Assay Kit has been optimized using purified PC-PLC
from Bacillus cereus. This assay may be amenable for use with cells and cell lysates, although the
presence of phospholipase A 2 or phospholipase D activity can potentially result in confound-
ing signal enhancement. Using the EnzChek® Direct Phospholipase C Assay Kit with purified
enzyme from Bacillus cereus, we can typically detect as little as 10 mU/mL PC-PLC after one
hour incubation at room temperature (Figure 17.4.18). This kit is also useful for characterizing
PC-PLC inhibition, and because it offers a direct measurement, the potential for false positives
in a compound screen is reduced. Figure 17.4.17 Ribbon representation of the ADIFAB free
fatty acid indicator (A3880). In the left-hand image, the
Each EnzChek® Direct Phospholipase C Assay Kit (2-plate size, E10215; 10-plate size, E10216) fatty acid binding site of intestinal fatty acid–binding pro-
provides: tein (yellow) is occupied by a covalently attached acrylodan
fluorophore (blue). In the right-hand image, a fatty acid
molecule (gray) binds to the protein, displacing the fluoro-
• Phosphatidylcholine-specific phospholipase C (PC-PLC) substrate phore (green) and producing a shift of its fluorescence emis-
• Phospholipase C from Bacillus cereus sion spectrum. Image contributed by Alan Kleinfeld, FFA
• Concentrated phospholipase C reaction buffer Sciences LLC, San Diego.
• Phosphatidylcholine (lecithin)
• Dimethylsulfoxide (DMSO)
• Detailed assay protocols
1,000
The 2-plate assay kit provides sufficient reagents for 200 reactions in 96-well microplates at a
volume of 200 µL per well or 2000 reactions using low-volume 384-well microplates at a volume 800
Fluorescence

of 20 µL per well. The 10-plate assay kit provides sufficient reagents for 1000 reactions in 96-well
600
microplates at a volume of 200 µL per well or 10,000 reactions using low-volume 384-well mi- 600
R2 = 0.9986

croplates at a volume of 20 µL per well. 400 400

200

Amplex® Red Phosphatidylcholine-Specific Phospholipase C Assay Kit 200

0
0 50 100 150
The Amplex® Red Phosphatidylcholine-Specific Phospholipase C Assay Kit (A12218) pro-
0
vides a sensitive method for continuously monitoring phosphatidylcholine-specific phospho- 0 100 200 300 400 500

lipase C (PC-PLC) activity in vitro using a fluorescence microplate reader or fluorometer.38–40 PLC concentration (mU/mL)
In this enzyme-coupled assay, PC-PLC activity is monitored indirectly using the Amplex® Red Figure 17.4.18 Detection of phosphatidylcholine-spe-
reagent, a sensitive fluorogenic probe for H2O2 (Section 10.5). First, PC-PLC converts the phos- cific phospholipase C (PC-PLC) using the EnzChek® Direct
Phospholipase C Assay Kit (E10215, E10216). Triplicate
phatidylcholine (lecithin) substrate to form phosphocholine and diacylglycerol. After the action samples of PC-PLC from Bacillus cereus were assayed at
of alkaline phosphatase, which hydrolyzes phosphocholine to inorganic phosphate and choline, concentration of 7.8 mU/mL to 500 mU/mL per well in the
choline is oxidized by choline oxidase to betaine and H2O2. Finally, H2O2, in the presence of presence of 1X PLC substrate and 200 μM lecithin in 1X PLC
reaction buffer. Reactions were incubated at room tem-
horseradish peroxidase, reacts with the Amplex® Red reagent in a 1:1 stoichiometry to generate
perature for 60 minutes and fluorescence was measured
the highly fluorescent product, resorufin. Because resorufin has absorption and fluorescence using excitation/emission wavelengths of 490/520 nm. The
emission maxima of approximately 571 nm and 585 nm, respectively, there is little interference inset represents a separate experiment and illustrates
the linearity of fluorescence response at low levels of PC-
from autofluorescence in most biological samples.
PLC. The average variation of replicates (CV) was less than
The Amplex® Red Phosphatidylcholine-Specific Phospholipase C Assay Kit is potentially useful 3%. Background fluorescence determined for the no-
for detecting PC-PLC activity in cell extracts and for screening PC-PLC inhibitors. Experiments enzyme control reaction has been subtracted.

The
TheMolecular
MolecularProbes Handbook:
Probes®
™
A Guide
Handbook: to Fluorescent
A Guide Probes
to Fluorescent and
Probes Labeling
and Technologies
Labeling Technologies
IMPORTANT NOTICE:described
The products described
manualinare
this covered
manual are
by covered by oneLimited
or moreUse
Limited Use Label License(s).
PleasePlease
referrefer to the Appendix
on on
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page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. 795
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 Chapter 17 — Probes for Signal Transduction Section 17.4 Probes for Lipid Metabolism and Signaling

with purified PC-PLC from Bacillus cereus indicate that the Amplex® Red Phosphatidylcholine-
1200 Specific Phospholipase C Assay Kit can detect PC-PLC levels as low as 0.2 mU/mL using a reaction
time of one hour (Figure 17.4.19). One unit of PC-PLC is defined as the amount of enzyme that
1000
will liberate 1.0 micromole of water-soluble organic phosphorus from L-α-phosphatidylcholine
per minute at pH 7.3 at 37°C.
Fluorescence

800

100
Each Amplex® Red Phosphatidylcholine-Specific Phospholipase C Assay Kit includes:
600
80

400 60 • Amplex® Red reagent

40
• Dimethylsulfoxide (DMSO)
200 20

0
• Horseradish peroxidase (HRP)
• H2O2 for use as a positive control
0 0.5 1 1.5 2
0
0 20 40 60 80 100 120
• Concentrated reaction buffer
PC-PLC (mU/mL)
• Choline oxidase from Alcaligenes sp.
Figure 17.4.19 Detection of phosphatidylcholine- • Alkaline phosphatase from calf intestine
specific phospholipase C using the Amplex® Red
Phosphatidylcholine-Specific Phospholipase C Assay Kit
• L-α-Phosphatidylcholine (lecithin)
(A12218). Fluorescence was measured in a fluorescence mi- • Phosphatidylcholine-specific phospholipase C from Bacillus cereus
croplate reader using excitation at 560 ± 10 nm and fluores- • Detailed protocols
cence detection at 590 ± 10 nm. The inset shows the sensitiv-
ity at very low enzyme concentrations.
Each kit provides sufficient reagents for approximately 500 assays using a fluorescence mi-
croplate reader and a reaction volume of 200 µL per assay.

Bacillus cereus PI-PLC

Phosphatidylinositol-specific phospholipase C (PI-PLC, EC [Link]) from Bacillus cereus
2400 cleaves phosphatidylinositol (PI), yielding water-soluble D-myo-inositol 1,2-cyclic monophos-
phate and lipid-soluble diacylglycerol.41 This enzyme also functions to release enzymes that are
2000
linked to glycosylphosphatidylinositol (GPI) membrane anchors. We offer highly purified B. ce-
reus PI-PLC (P6466), which has been used in studies of PI synthesis and export across the plasma
Fluorescence

1600

800 membrane.42 PI-PLC generates diacylglycerols for PKC-linked signal transduction studies 43 and
1200
provides an efficient means of releasing most GPI-anchored proteins from cell surfaces under
800
400 conditions in which the cells remain viable.44,45
400
0

Phospholipase D Assays
0 5 10 15 20 25
0
0 100 200 300 400
Phospholipase D (mU/mL)
Amplex® Red Phospholipase D Assay Kit
Figure 17.4.20 Quantitation of phospholipase D from The Amplex® Red Phospholipase D Assay Kit (A12219) provides a sensitive method for mea-
Streptomyces chromofuscus using the Amplex® Red
Phospholipase D Assay Kit (A12219). Fluorescence was mea-
suring phospholipase D (PLD) activity in vitro using a fluorescence microplate reader or fluorom-
sured with a fluorescence microplate reader using excita- eter.38,40,46 In this enzyme-coupled assay, PLD activity is monitored indirectly using the Amplex®
tion at 530 ± 12.5 nm and fluorescence detection at 590 ± Red reagent (Section 10.5). First, PLD cleaves the phosphatidylcholine (lecithin) substrate to
17.5 nm. The inset shows the sensitivity at very low enzyme
concentrations (0–25 mU/mL).
yield choline and phosphatidic acid. Second, choline is oxidized by choline oxidase to betaine
and H2O2. Finally, H2O2, in the presence of horseradish peroxidase, reacts with the Amplex®
Red reagent to generate the highly fluorescent product, resorufin (excitation/emission maxima
~571/585 nm).
The Amplex® Red Phospholipase D Assay Kit is designed for detecting PLD activity in cell ex-
tracts and for screening PLD [Link] kit can be used to continuously assay PLD enzymes
with near-neutral pH optima, whereas PLD enzymes with acidic pH optima can be assayed in
N N
�
O a simple two-step procedure. Experiments with purified PLD from Streptomyces chromofuscus
H3C � � (CH2)�� C OCH2
indicate that the Amplex® Red Phospholipase D Assay Kit can detect PLD levels as low as 10 mU/
(CH3)2N N N C NH(CH2)�� C OCH
O O
mL using a reaction time of one hour (Figure 17.4.20). One unit of PLD is defined as the amount
CH2O(CH2)�CH3
of enzyme that will liberate 1.0 micromole of choline from L-α-phosphatidylcholine per minute
Figure 17.4.21 EnzChek® lipase substrate (E33955).
at pH 8.0 at 30°C. Each Amplex® Red Phospholipase D Assay Kit includes:

• Amplex® Red reagent • Concentrated reaction buffer

• Dimethylsulfoxide (DMSO) • Choline oxidase from Alcaligenes sp.
• Horseradish peroxidase (HRP) • L-α-Phosphatidylcholine (lecithin)
• H2O2 for use as a positive control • Detailed protocols

The
The MolecularProbes®
Molecular Probes Handbook:
Handbook: AA Guide
Guide to
to Fluorescent
™
Probesand
Fluorescent Probes andLabeling
LabelingTechnologies
Technologies
IMPORTANT NOTICE: The products described in this manual
in thisaremanual
coveredare
by covered
one or more Limited Use Label License(s). Please refer to thePlease
Appendix onto
796 IMPORTANT NOTICE : The products described by one or more Limited Use Label License(s).
the Appendix onrefer
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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Chapter 17 — Probes for Signal Transduction Section 17.4 Probes for Lipid Metabolism and Signaling

Each kit provides sufficient reagents for approximately 500 assays Anti-Phosphoinositide Monoclonal Antibodies
using a fluorescence microplate reader and a reaction volume of 200 µL
per assay. Phosphatidylinositol (PI or PtdIns) and its phosphorylated deriva-
tives represent only a small fraction of eukaryotic cellular phospholip-
Fluorescent Substrates for Phospholipase D ids but are functionally significant in a disproportionately large number
The products of phospholipase A 2, C and D cleavage of 1-O-alkyl- of regulatory and signal transduction processes.55–60 The most famil-
2-decanoyl-sn-glycero-3-phosphocholine labeled with the BODIPY® iar of these processes is the phospholipase C–mediated generation of
FL fluorophore (D3771, Figure 17.4.10) can be separated and indepen- the ubiquitous second messengers inositol 1,4,5-triphosphate (InsP3)
dently quantitated based on their differential migration on TLC or and diacylglycerol (DAG) from phosphatidylinositol 4,5-diphosphate
HPLC.23,47,48 Our BODIPY® FL analog is preferred for this application (PtdIns(4,5)P2; Section 17.2). Research has revealed the direct action
because it is relatively photostable and the fluorescence properties of of phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P2) and phospha-
its different enzymatic products are all very similar.49 Researchers have tidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3) on a diverse array of
taken advantage of these features to detect and quantitate phospholi- cellular functions, including actin assembly and cytoskeletal dynam-
pase D activity in vascular smooth muscle cells,49,50 cultured mamma- ics, vesicular protein trafficking, protein kinase localization and activa-
lian cells 51 and yeast.52–54 tion, cell proliferation and apoptosis. We offer mouse monoclonal IgM
antibodies to PtdIns(4,5)P2 (A21327) and PtdIns(3,4,5)P3 (A21328) for
immunocytochemical localization of these important lipid metabo-
EnzChek® Lipase Substrate lites.61 Both antibodies have been shown to recognize their cognate
phosphoinositides in murine and human cells with only slight cross-
The triacylglycerol-based EnzChek® lipase substrate (E33955, reactivity with other phosphoinositides or phospholipids.
Figure 17.4.21) offers higher throughput and better sensitivity than
chromogenic (TLC or HPLC) assays, and a visible light–excitable al-
ternative to 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMU oc- Sphingolipids
tanoate, D12200; Section 10.6). In the presence of lipases, the nonfluo-
rescent EnzChek® lipase substrate produces a bright, green-fluorescent Sphingolipids include sphingomyelins, which are phospholipid ana-
product (excitation/emission maxima of ~505/515 nm) for the accurate logs, as well as ceramides, glycosyl ceramides (cerebrosides), ganglio-
and sensitive detection of lipase activity in solution. sides and other derivatives (Figure 17.4.22). Several excellent reviews of
the chemistry and biology of sphingolipids and glycosphingolipids and
their role in the process of signal transduction are available.62,63
A HOCH CH CH
2 2
OH OH
Glycerol
O O
+ <
(CH ) NCH CH O P O CH CH CH O P O CH CH CH
33 2 2 2 2 2 2
< <
O O O O O O
O C C O O C C O
R R R R
OH OH O
Phosphatidylcholine Phosphatidic Acid
O P O CH CH CH
< 2 2
OH O O
O
HO O C C O
OH R R

Phosphatidylinositol

HOCH CH CH CH CH(CH ) CH
2 2 12 3
B NH OH
2

Sphingosine
O
+ HO
(CH ) NCH CH O P O CH CH CH CH CH(CH ) CH
33 2 2 2 2 12 3
<
NH OH O O CH 2 CH CH CH CH(CH ) CH
2 12 3
O OH NH OH
C O
HO C O
R OH
Sphingomyelin R
Cerebroside
HOCH CH CH CH CH(CH ) CH
2 2 12 3
NH OH
C O
R
Ceramide
Figure 17.4.22 A) Phosphatidylcholines, phosphatidylinositols and phosphatidic acids are examples of glycerolipids derived from glycerol. B) Sphingomyelins, ceramides
and cerebrosides are examples of sphingolipids derived from sphingosine. In all the structures shown, R represents the hydrocarbon tail portion of a fatty acid residue.

TheMolecular
The Molecular Probes®
Probes™
Handbook:
Handbook: A Guide
A Guide to Fluorescent
to Fluorescent Probes
Probes andand Labeling
Labeling Technologies
Technologies
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
IMPORTANT NOTICEpage: The products
971 and Masterdescribed
Product Listinon
this manual
page are covered
975. Products are Forby one or Use
Research more Limited
Only. Use Label
Not intended License(s).
for any animal orPlease refer to theorAppendix
human therapeutic onuse.
diagnostic 797
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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 Chapter 17 — Probes for Signal Transduction Section 17.4 Probes for Lipid Metabolism and Signaling

BODIPY® Sphingolipids
Ceramides (N-acylsphingosines), like diacylglycerols, are lipid second messengers that func-
tion in signal transduction processes.63–65 The concentration-dependent spectral properties of
BODIPY® FL C5-ceramide (D3521, B22650; Figure 17.4.23), BODIPY® FL C5-sphingomyelin 66–68
(D3522, Figure 17.4.24) and BODIPY® FL C12-sphingomyelin 69 (D7711) make them particularly
suitable for investigating sphingolipid transport and metabolism,68,70–73 in addition to their appli-
Figure 17.4.23 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- cations as structural markers for the Golgi complex 74 (Section 12.4). BODIPY® FL C5-ceramide
diaza-s-indacene-3-pentanoyl)sphingosine (BODIPY® FL C5- can be visualized by fluorescence microscopy 75,76 (Figure 17.4.25, Figure 17.4.26) or by electron
ceramide) (D3521).
microscopy following diaminobenzidine (DAB) photoconversion to an electron-dense product.77
(Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2).
Our range of BODIPY® sphingolipids also includes the long-wavelength light–ex-
citable BODIPY® TR ceramide 78,79 (D7540, Figure 17.4.27), as well as BODIPY® FL C5-
lactosylceramide 80–85 (D13951), BODIPY® FL C5-ganglioside GM186 (B13950, Figure 17.4.28)
and BODIPY® FL C12-galactocerebroside (D7519). All of our sphingolipids are prepared from
D-erythro-sphingosine and therefore have the same stereochemical conformation as natural
Figure 17.4.24 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- biologically active sphingolipids.87
diaza-s-indacene-3-pentanoyl)sphingosyl phosphocholine Complexing fluorescent lipids with defatted bovine serum albumin (BSA) facilitates cell
(BODIPY® FL C5-sphingomyelin) (D3522).
labeling by eliminating the need for organic solvents to dissolve the lipophilic probe; the

Figure 17.4.25 Nucleus and Golgi apparatus of a bovine

pulmonary artery endothelial cell (BPAEC) labeled with
Hoechst 33342 (H1399, H3569, H21492) and the BSA com-
plex of BODIPY® FL C5-ceramide (B22650), respectively.

Figure 17.4.28 BODIPY® FL C5-ganglioside GM1 (B13950).

Figure 17.4.26 Cells in the notochord rudiment of a ze-

brafish embryo undergoing mediolateral intercalation to
lengthen the forming notochord. BODIPY® FL C5-ceramide
(D3521) localizes in the interstitial fluid of the zebrafish
embryo and freely diffuses between cells, illuminating cell
boundaries. This confocal image was obtained using a Bio-
Rad® MRC-600 microscope. Image contributed by Mark
Cooper, University of Washington.

Figure 17.4.29 Selective staining of the Golgi appara- Figure 17.4.30 Live J774 macrophage cells labeled
tus using the green-fluorescent BODIPY® FL C5-ceramide with BODIPY® FL C5-ganglioside GM1 (B13950) and then
(D3521) (top panel). At high concentrations, the BODIPY® with Alexa Fluor® 555 cholera toxin subunit B conjugate
FL fluorophore forms excimers that can be visualized using (C22843). Cells were then treated with anti–CT-B antibody
a red longpass optical filter (bottom panel). The BODIPY® FL to induce crosslinking. Yellow fluorescence indicates colo-
Figure 17.4.27 BODIPY® TR ceramide (N-((4-(4,4-difluoro- C5-ceramide accumulation in the trans-Golgi is sufficient for calization of the two dyes. Nuclei were stained with the blue
5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy) excimer formation (J Cell Biol (1991) 113:1267). Images con- fluorescent Hoechst 33342 dye (H1399, H3570, H21492).
acetyl)sphingosine) (D7540). tributed by Richard Pagano, Mayo Foundation.

The
TheMolecular
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
Guideto
™
toFluorescent
Fluorescent Probes
Probes and
and Labeling
LabelingTechnologies
Technologies
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
798 IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer tothe Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
[Link]/probes
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Chapter 17 — Probes for Signal Transduction Section 17.4 Probes for Lipid Metabolism and Signaling

BSA-complexed probe can be directly dissolved in water.88 We offer four BODIPY® sphingo-
lipid–BSA complexes for the study of lipid metabolism and trafficking, including:

• BODIPY® FL C5-ceramide (B22650)

• BODIPY® TR ceramide (B34400)
• BODIPY® FL C5-lactosylceramide (B34401)
• BODIPY® FL C5-ganglioside GM1 (B34402) Figure 17.4.32 NBD C6-ceramide (6-((N-(7-nitrobenz-2-oxa-
1,3-diazol-4-yl)amino)hexanoyl)sphingosine) (N1154).
BODIPY® FL C5-ceramide has been used to investigate the linkage of sphingolipid metabo-
lism to protein secretory pathways 89–91 and neuronal growth.83,92 Internalization of BODIPY® O O
FL C5-sphingomyelin (D3522) from the plasma membrane of human skin fibroblasts results in NH(CH )
25
C OH NH(CH )
25
C OH
a mixed population of labeled endosomes that can be distinguished based on the concentration- N S O
2− N
2 4
dependent green (~515 nm) or red (~620 nm) emission of the probe 68 (Figure 17.4.29). BODIPY® O O
N N
C5-sphingomyelin has also been used to assess sphingomyelinase gene transfer and expression in
NO NH
hematopoietic stem and progenitor cells.93 BODIPY® FL C5-lactosylceramide, BODIPY® FL C5- 2 2

ganglioside GM1 and BODIPY® FL cerebrosides are useful tools for the study of glycosphingolipid Fluorescent Nonfluorescent
transport and signaling pathways in cells.94–97 BODIPY® FL C5-ganglioside GM1 has been shown to Figure 17.4.33 Dithionite reduction of 6-(N-(7-nitrobenz-2-
oxa-1,3-diazol-4-yl)amino)hexanoic acid (NBD-X, N316). The
form cholesterol-enhanced clusters in membrane complexes with amyloid β-protein in a model of
elimination of fluorescence associated with this reaction,
Alzheimer disease amyloid fibrils.98 Colocalization of fluorescent cholera toxin B conjugates (Section coupled with the fact that extraneously added dithionite is
7.7) and BODIPY® FL C5-ganglioside GM1 observed by fluorescence microscopy provides a direct not membrane permeant, can be used to determine wheth-
er the NBD fluorophore is located in the external or internal
indication of the association of these molecules in lipid rafts 99,100 (Figure 17.4.30, Figure 17.4.31).
monolayer of lipid bilayer membranes.

NBD Sphingolipids
NBD C6 -ceramide (N1154, Figure 17.4.32) and NBD C6 -sphingomyelin (N3524) analogs 6,000
predate their BODIPY® counterparts and have been extensively used for following sphingolipid
metabolism in cells 101–103 and in multicellular organisms.104 As with BODIPY® FL C5-ceramide, 5,000

we also offer NBD C6 -ceramide complexed with defatted BSA (N22651) to facilitate cell loading

Fluorescence
4,000

without the use of organic solvents to dissolve the probe.88 Elimination of NBD fluorescence at 1,000
3,000
the extracellular surface by dithionite reduction (Figure 17.4.33) can be used to assess endocyto- 800

sis and recycling of NBD sphingolipids. 2,000 600

400

1,000 200

Amplex® Red Sphingomyelinase Assay Kit 0

0 0.2 0.4 0.6
The Amplex® Red Sphingomyelinase Assay Kit (A12220) is designed for measuring sphin- 0
0 10 20 30 40 50
gomyelinase activity in solution using a fluorescence microplate reader or fluorometer (Figure Sphingomyelinase (mU/mL)
17.4.34). This assay should be useful for screening sphingomyelinase activators or inhibitors or Figure 17.4.34 Measurement of sphingomyelinase activ-
for detecting sphingomyelinase activity in cell and tissue extracts. The assay, which uses natural ity using the Amplex® Red Sphingomyelinase Assay Kit
sphingomyelin as the principal substrate, employs an enzyme-coupled detection scheme in which (A12220). Each reaction contained 50 µM Amplex® Red re-
agent, 1 U/mL horseradish peroxidase (HRP), 0.1 U/mL cho-
phosphocholine liberated by the action of sphingomyelinase is cleaved by alkaline phosphatase line oxidase, 4 U/mL of alkaline phosphatase, 0.25 mM sphin-
to generate choline. Choline is, in turn, oxidized to betaine by choline oxidase, generating H 2O2, gomyelin and the indicated amount of Staphylococcus aureus
which drives the conversion of the Amplex® Red reagent (A12222, A22177; Section 10.5) to red- sphingomyelinase in 1X reaction buffer. Reactions were in-
cubated at 37°C for 1 hour. Fluorescence was measured with
fluorescent resorufin (Figure 17.4.35). This sensitive assay technique has been employed to detect a fluorescence microplate reader using excitation at 530 ±
activation of acid sphingomyelinase associated with ultraviolet radiation–induced apoptosis 105 12.5 nm and fluorescence detection at 590 ± 17.5 nm.
and to characterize an insecticidal sphingomyelinase C produced by Bacillus cereus.106

Figure 17.4.31. A J774 mouse macrophage cell sequentially stained with BODIPY® FL ganglioside GM1 (B13950) and then with
Alexa Fluor® 555 dye–labeled cholera toxin subunit B (C22843, C34776). The cell was then treated with an anti–CT-B antibody
to induce crosslinking. Alexa Fluor® 555 dye fluorescence (left panel, red) and BODIPY® FL dye fluorescence (middle panel, Figure 17.4.35 Absorption and fluorescence emission
green) were imaged separately and overlaid to emphasize the coincident staining (right panel, yellow). Nuclei were stained spectra of resorufin in pH 9.0 buffer.
with blue-fluorescent Hoechst 33258 (H1398, H3569, H21491).

The
TheMolecular
MolecularProbes Handbook:
Probes®
™
A Guide
Handbook: to Fluorescent
A Guide Probes
to Fluorescent and
Probes Labeling
and Technologies
Labeling Technologies
IMPORTANT NOTICE:described
The products described
manualinare
this covered
manual are
by covered by oneLimited
or moreUse
Limited Use Label License(s).
PleasePlease
referrefer to the Appendix
on on
IMPORTANT NOTICE : The products in this one or more Label License(s). to the Appendix
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. 799
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
[Link]/probes
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 Chapter 17 — Probes for Signal Transduction Section 17.4 Probes for Lipid Metabolism and Signaling

The Amplex® Red Sphingomyelinase Assay Kit contains:

Ex = 390 nm
_OA
• Amplex® Red reagent
Fluorescence emission

• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
+OA
• H2O2 for use as a positive control
• Concentrated reaction buffer
• Choline oxidase from Alcaligenes sp.
• Alkaline phosphatase from calf intestine
• Sphingomyelin
• Triton X-100
400 450 500 550 600 650
• Sphingomyelinase from Bacillus sp.
Wavelength (nm)
• Detailed protocols
Figure 17.4.36 The free fatty acid–dependent spectral
shift of ADIFAB reagent (A3880). Spectra shown represent
0.2 µM ADIFAB in pH 8.0 buffer with (+OA) and without Each kit provides sufficient reagents for approximately 500 assays using a fluorescence mi-
(–OA) addition of 4.7 µM cis-9-octadecenoic (oleic) acid croplate reader and a reaction volume of 200 µL per assay.
(OA). The ratio of fluorescence emission intensities at 505 nm
and 432 nm can be quantitatively related to free fatty acid
concentrations.
ADIFAB Reagent: A Unique Free Fatty Acid Indicator
Elevated levels of free fatty acids (FFA)—which are associated with multiple pathological states,
including cancer, diabetes and cardiac ischemia 107—are generated by inflammatory responses,
phospholipase A activity and cytotoxic phenomena.108 Sensitive techniques are required to detect
and quantitate free fatty acids because these important metabolites have low aqueous solubility and
are usually found complexed to carriers. ADIFAB (A3880) is a dual-wavelength fluorescent FFA
indicator that consists of a polarity-sensitive fluorescent probe (acrylodan, A433; Section 2.3) con-
jugated to I-FABP, a rat intestinal fatty acid–binding protein with a low molecular weight (15,000
daltons) and a high binding affinity for FFA 109–111 (Figure 17.4.17).
As shown in Figure 17.4.36, titration of the ADIFAB reagent with oleic acid results in a shift of its
fluorescence maximum from ~432 nm to ~505 nm. The ratio (R) of these signals (505 nm/432 nm)
can be converted to an FFA concentration by using the FFA dissociation constant (Kd) and employ-
ing analysis procedures similar to those developed for Ca2+ indicators 112 (Chapter 19). Values of Kd
vary considerably for different fatty acids; a typical value is 0.28 µM for oleic acid 111 (determined
at 37°C). There is little, if any, interference from bile acids, glycerides, sterols or bilirubin. With
appropriate precautions, which are described in the product information sheet accompanying this
product, ADIFAB can be used to determine FFA concentrations in the range 1 nM to >20 µM.
ADIFAB was used to investigate the physical basis of cis-unsaturated fatty acid inhibition of
cytotoxic T cells.113 This effect is due to inhibition of a specific tyrosine phosphorylation event
that normally accompanies antigen stimulation.114,115 Measurements using ADIFAB have also
revealed previously undetected differences in FFA binding affinities among fatty acid–binding
proteins from different tissues 116,117 and have enabled quantitation of FFA levels in human serum
as a potential diagnostic tool.107,118

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1. Nat Rev Mol Cell Biol (2001) 2:327; 2. Curr Opin Struct Biol (2000) 10:737; 3. Physiol Rev (1999) 38:3867; 36. Anal Biochem (1995) 229:256; 37. Biochem J (1994) 298:23; 38. Proc
(2000) 80:1291; 4. Biochim Biophys Acta (1994) 1212:26; 5. Biochim Biophys Acta (2000) Natl Acad Sci U S A (2004) 101:9745; 39. Mol Pharmacol (2000) 57:1142; 40. Proc SPIE-
1488:124; 6. Mol Med Today (1999) 5:244; 7. FASEB J (1994) 8:916; 8. Cardiovasc Drugs Int Soc Opt Eng (2000) 3926:166; 41. J Mol Biol (1998) 275:635; 42. Biochim Biophys
Ther (2009) 23:49; 9. Clin Chim Acta (2010) 411:190; 10. Methods Enzymol (2007) 434:15; Acta (1994) 1224:247; 43. J Biol Chem (1994) 269:4098; 44. J Biol Chem (1991) 266:1926;
11. Circ Res (2009) 104:952; 12. Am J Physiol Gastrointest Liver Physiol (2009) 296:G445; 45. J Neurochem (1991) 57:67; 46. J Biol Chem (2002) 277:45592; 47. Anal Biochem (2000)
13. Science (2001) 292:1385; 14. Sci Signal (2009) 2:ra71-ra71; 15. J Invest Dermatol 286:277; 48. J Biol Chem (1997) 272:12909; 49. Anal Biochem (1994) 218:136; 50. J Biol
(2009) 129:2772; 16. Anal Biochem (1999) 276:27; 17. Science (2000) 288:1160; 18. Br Chem (1994) 269:23790; 51. Mol Biol Cell (1999) 10:3863; 52. Eukaryot Cell (2009) 8:1094;
J Pharmacol (1998) 124:1675; 19. J Biol Chem (1992) 267:21465; 20. J Biol Chem (2001) 53. Biochem J (1996) 314:15; 54. Biochem J (1995) 307:799; 55. Biochem J (2001) 360:513;
276:12035; 21. J Lipid Res (2007) 48:385; 22. J Lipid Res (2007) 48:472; 23. Anal Biochem 56. Biochem J (2001) 355:249; 57. Cell (2000) 100:603; 58. J Biol Chem (1999) 274:8347;
(1990) 185:80; 24. J Biol Chem (1999) 274:19338; 25. Anal Biochem (1981) 116:553; 59. J Biol Chem (1999) 274:9907; 60. Annu Rev Cell Dev Biol (1998) 14:231; 61. J Histochem
26. Biochemistry (1995) 34:2049; 27. Biochim Biophys Acta (2000) 1486:321; 28. J Lipid Cytochem (2002) 50:697; 62. Science (2010) 327:46; 63. Biochemistry (2001) 40:4893;
Res (1992) 33:1863; 29. J Neurosci Methods (2000) 100:127; 30. Biochemistry (1999) 64. Trends Cell Biol (2000) 10:408; 65. J Biol Chem (1994) 269:3125; 66. Chem Phys Lipids
38:7803; 31. Biochemistry (1997) 36:14325; 32. Biochim Biophys Acta (1987) 917:411; (1999) 102:55; 67. Ann N Y Acad Sci (1998) 845:152; 68. Biophys J (1997) 72:37; 69. J Cell
33. J Biol Chem (2001) 276:22732; 34. J Biol Chem (1999) 274:11494; 35. Biochemistry Biol (1998) 140:39; 70. Methods Enzymol (2000) 312:293; 71. Methods Enzymol (2000)

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Chapter 17 — Probes for Signal Transduction Section 17.4 Probes for Lipid Metabolism and Signaling

REFERENCES—continued
312:523; 72. Frontiers in Bioactive Lipids, Vanderhoek JV, Ed. 1996, p 203; 73. J Cell Biol Cell (2007) 18:2667; 96. J Cell Biol (2007) 176:895; 97. Methods (2005) 36:186; 98. J Biol
(1996) 134:1031; 74. J Cell Biol (1991) 113:1267; 75. Cytometry (1993) 14:251; 76. J Cell Chem (2001) 276:24985; 99. Mol Biol Cell (2009) 20:3751; 100. J Cell Sci (2009) 122:289;
Biol (1993) 120:399; 77. Eur J Cell Biol (1992) 58:214; 78. Mol Biochem Parasitol (2000) 101. Biochim Biophys Acta (1992) 1113:277; 102. Adv Cell Mol Biol Membranes (1993)
106:21; 79. Infect Immun (2000) 68:5960; 80. J Cell Biol (2001) 154:535; 81. Am J Physiol 1:199; 103. Biochim Biophys Acta (1991) 1082:113; 104. Parasitology (1992) 105:81;
Lung Cell Mol Physiol (2001) 280:L938; 82. Nat Cell Biol (1999) 1:386; 83. J Neurochem 105. J Biol Chem (2001) 276:11775; 106. Eur J Biochem (2004) 271:601; 107. Am J Cardiol
(1999) 73:1375; 84. Lancet (1999) 354:901; 85. Proc Natl Acad Sci U S A (1998) 95:6373; (1996) 78:1350; 108. J Immunol (1991) 147:2809; 109. J Biol Chem (1995) 270:15076;
86. J Cell Biol (1999) 147:447; 87. Biophys J (1999) 77:1498; 88. Cell Biology: A Laboratory 110. Biochemistry (1993) 32:7574; 111. J Biol Chem (1992) 267:23495; 112. Mol Cell
Handbook, 2nd Ed., Vol. 2, Celis JE, Ed. 1998, p. 507; 89. Mol Biol Cell (1995) 6:135; Biochem (1999) 192:87; 113. Biochemistry (1993) 32:530; 114. J Biol Chem (1994) 269:9506;
90. J Biol Chem (1993) 268:4577; 91. Biochemistry (1992) 31:3581; 92. J Biol Chem 115. J Biol Chem (1993) 268:17578; 116. Biochemistry (2000) 39:7197; 117. J Biol Chem
(1993) 268:14476; 93. Blood (1999) 93:80; 94. J Immunol (2007) 179:6770; 95. Mol Biol (1994) 269:23918; 118. J Lipid Res (1995) 36:229.

DATA TABLE 17.4 PROBES FOR LIPID METABOLISM AND SIGNALING

Cat. No. MW Storage Soluble Abs EC Em Solvent Notes
A3880 ~15,350 FF,L,AA H2O 365 10,500 432 H2O 1
A10070 880.68 FF,D,L DMSO 505 92,000 512 MeOH 2, 16
A10072 986.67 FF,D,L DMSO 505 85,000 567 MeOH 2, 17, 18
B3781 797.88 FF,D,L see Notes 342 75,000 471 EtOH 2, 3
B3782 966.20 FF,D,L see Notes 340 62,000 473 EtOH 2, 4
B7701 1029.80 FF,D,L see Notes 505 123,000 512 MeOH 2, 5
B13950 1582.50 F,D,L DMSO, EtOH 505 80,000 512 MeOH 6
B22650 ~66,000 F,D,L H2O 505 91,000 511 MeOH 6, 7
B34400 ~66,000 F,D,L H2O 589 65,000 616 MeOH 7
B34401 ~66,000 F,D,L H2O 505 80,000 512 MeOH 6, 7
B34402 ~66,000 F,D,L H2O 505 80,000 511 MeOH 6, 7
D3521 601.63 FF,D,L CHCl3, DMSO 505 91,000 511 MeOH 6
D3522 766.75 FF,D,L see Notes 505 77,000 512 MeOH 2, 6
D3771 854.86 FF,D,L see Notes 506 71,000 512 EtOH 2
D3803 797.77 FF,D,L see Notes 503 80,000 512 MeOH 2, 8
D7519 861.96 FF,D,L DMSO, EtOH 505 85,000 511 MeOH 6
D7540 705.71 FF,D,L CHCl3, DMSO 589 65,000 616 MeOH
D7711 864.94 FF,D,L DMSO 505 75,000 513 MeOH 6, 9
D13951 925.91 FF,D,L DMSO, EtOH 505 80,000 511 MeOH 6
D23739 1136.13 FF,D,L DMSO 505 92,000 511 MeOH 2, 10
E33955 1011.15 F,D,L CHCl3 DMSO 505 94,000 515 MeOH 11
H361 850.13 FF,D,L see Notes 342 37,000 376 MeOH 2, 12, 13
H3809 856.09 FF,D,L see Notes 341 38,000 376 MeOH 2, 12, 13
N1154 575.75 FF,D,L CHCl3, DMSO 466 22,000 536 MeOH 14
N3524 740.88 FF,D,L see Notes 466 22,000 536 MeOH 2, 14
N3786 771.89 FF,D,L see Notes 465 21,000 533 EtOH 2, 14, 15
N3787 856.05 FF,D,L see Notes 465 22,000 534 EtOH 2, 14, 15
N22651 ~66,000 F,D,L H2O 466 22,000 536 MeOH 7, 14
For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.
Notes
1. ADIFAB fatty acid indicator is a protein conjugate with a molecular weight of approximately 15,350. Em shifts from about 432 nm to 505 nm upon binding of fatty acids. (Mol Cell Biochem (1999)
192:87)
2. Chloroform is the most generally useful solvent for preparing stock solutions of phospholipids (including sphingomyelins). Glycerophosphocholines are usually freely soluble in ethanol. Most
other glycerophospholipids (phosphoethanolamines, phosphatidic acids and phosphoglycerols) are less soluble in ethanol, but solutions up to 1–2 mg/mL should be obtainable, using sonica-
tion to aid dispersion if necessary. Labeling of cells with fluorescent phospholipids can be enhanced by addition of cyclodextrins during incubation. (J Biol Chem (1999) 274:35359)
3. Phospholipase A cleavage generates a fluorescent fatty acid (P1903MP (Section 13.2)) and a fluorescent lysophospholipid.
4. Phospholipase A cleavage generates a fluorescent fatty acid (P31 (Section 13.2)) and a fluorescent lysophospholipid.
5. Phospholipase A cleavage results in increased fluorescence with essentially no wavelength shift. The cleavage products are D3862 (Section 13.2) and a fluorescent lysophospholipid.
6. Em for BODIPY® FL sphingolipids shifts to ~620 nm when high concentrations of the probe (>5 mol %) are incorporated in lipid mixtures. (J Cell Biol (1991) 113:1267)
7. This product is a lipid complexed with bovine serum albumin (BSA). Spectroscopic data are for the free lipid in MeOH.
8. Phospholipase A2 cleavage generates a fluorescent fatty acid (D3834 (Section 13.2)) and a nonfluorescent lysophospholipid.
9. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."
10. Phospholipase A2 cleavage results in increased fluorescence with essentially no wavelength shift. The cleavage products are D3834 (Section 13.2) and a dinitrophenylated lysophospholipid.
11. Fluorescence of the intact substrate is weak. Lipase hydrolysis releases a highly fluorescent fatty acid (D3823, Section 13.2).
12. Pyrene derivatives exhibit structured spectra. The absorption maximum is usually about 340 nm with a subsidiary peak at about 325 nm. There are also strong absorption peaks below
300 nm. The emission maximum is usually about 376 nm with a subsidiary peak at 396 nm. Excimer emission at about 470 nm may be observed at high concentrations.
13. Phospholipase A2 hydrolysis releases a fluorescent fatty acid; P31 (Section 13.2).
14. Fluorescence of NBD and its derivatives in water is relatively weak. QY and τ increase and Em decreases in aprotic solvents and other nonpolar environments relative to water. (Biochemistry
(1977) 16:5150, Photochem Photobiol (1991) 54:361)
15. Phospholipase A2 hydrolysis releases a fluorescent fatty acid; N316 (Section 13.2) from N3786 or N678 (Section 13.2) from N3787.
16. Phospholipase A1 cleavage results in increased fluorescence with essentially no wavelength shift. The cleavage products are D3834 (Section 13.2) and a dinitrophenylated lysophospholipid.
17. A10072 exhibits dual emission (Em = 510 nm and 567 nm in MeOH, 513 nm and 575 nm when incorporated in phospholipid bilayer membranes). Phospholipase A2 cleavage results in in-
creased 510–513 nm emission and reciprocally diminshed 567–575 nm emission.
18. A10072 is also soluble at 2 mM in 2-methoxyethanol.

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Chapter 17 — Probes for Signal Transduction Section 17.4 Probes for Lipid Metabolism and Signaling

PRODUCT LIST 17.4 PROBES FOR LIPID METABOLISM AND SIGNALING

Cat. No. Product Quantity
A3880 ADIFAB fatty acid indicator 200 µg
A12218 Amplex® Red Phosphatidylcholine-Specific Phospholipase C Assay Kit *500 assays* 1 kit
A12219 Amplex® Red Phospholipase D Assay Kit *500 assays* 1 kit
A12220 Amplex® Red Sphingomyelinase Assay Kit *500 assays* 1 kit
A21328 anti-phosphatidylinositol 3,4,5-triphosphate, mouse IgM, monoclonal RC6F8 (anti-PtdIns(3,4,5)P3) *1 mg/mL* 100 µL
A21327 anti-phosphatidylinositol 4,5-diphosphate, mouse IgM, monoclonal 2C11 (anti-PtdIns(4,5)P2) *1 mg/mL* 100 µL
B3781 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine 1 mg
B3782 1,2-bis-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine 1 mg
B7701 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine (bis-BODIPY® FL C11-PC) 100 µg
B22650 BODIPY® FL C5-ceramide complexed to BSA 5 mg
B13950 BODIPY® FL C5-ganglioside GM1 25 µg
B34401 BODIPY® FL C5-ganglioside GM1 complexed to BSA 1 mg
B34402 BODIPY® FL C5-lactosylceramide complexed to BSA 1 mg
B34353 BODIPY® FL phosphatidylinositol(4,5) bisphosphate (BODIPY® FL PtdIns(4,5)P2) 50 µg
D7540 BODIPY® TR ceramide (N-((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)sphingosine) 250 µg
B34400 BODIPY® TR ceramide complexed to BSA 5 mg
D3771 2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)undecyl)-sn-glycero-3-phosphocholine 1 mg
D7519 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)sphingosyl 1-β-D-galactopyranoside (BODIPY® FL C12-galactocerebroside) 25 µg
D7711 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)sphingosyl phosphocholine (BODIPY® FL C12-sphingomyelin) *1 mg/mL in DMSO* 250 µL
D3803 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® FL C5-HPC) 100 µg
D3521 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosine (BODIPY® FL C5-ceramide) 250 µg
D13951 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosyl 1-β-D-lactoside (BODIPY® FL C5-lactosylceramide) 25 µg
D3522 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosyl phosphocholine (BODIPY® FL C5-sphingomyelin) 250 µg
D23739 N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3- 1 mg
phosphoethanolamine, triethylammonium salt (PED6)
E10215 EnzChek® Direct Phospholipase C Assay Kit *phosphatidylcholine specific* *2-plate size* 1 kit
E10216 EnzChek® Direct Phospholipase C Assay Kit *phosphatidylcholine specific* *10-plate size* 1 kit
E33955 EnzChek® lipase substrate *green fluorescent, 505/515* 100 µg
E10219 EnzChek® Phospholipase A1 Assay Kit *2-plate size* 1 kit
E10221 EnzChek® Phospholipase A1 Assay Kit *10-plate size* 1 kit
E10217 EnzChek® Phospholipase A2 Assay Kit *2-plate size* 1 kit
E10218 EnzChek® Phospholipase A2 Assay Kit *10-plate size* 1 kit
H361 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (β-py-C10-HPC) 1 mg
H3809 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol, ammonium salt (β-py-C10-PG) 1 mg
N1154 NBD C6-ceramide (6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine) 1 mg
N22651 NBD C6-ceramide complexed to BSA 5 mg
N3524 NBD C6-sphingomyelin (6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosyl phosphocholine) 1 mg
N3787 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD C12-HPC) 5 mg
N3786 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD C6-HPC) 5 mg
A10070 PED-A1 (N-((6-(2,4-DNP)amino)hexanoyl)-1-(BODIPY® FL C5)-2-hexyl-sn-glycero-3-phosphoethanolamine) *phospholipase A1 selective substrate* 100 µg
P6466 phospholipase C, phosphatidylinositol-specific *from Bacillus cereus* *100 U/mL* 50 µL
A10072 Red/Green BODIPY® PC-A2 (1-O-(6-BODIPY® 558/568-aminohexyl)-2-BODIPY® FL C5-sn-glycero-3-phosphocholine) *ratiometric phospholipase A2 substrate* 100 µg

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Probes Handbook:
Handbook: AAGuide
Guide to
to Fluorescent Probesand
Fluorescent Probes andLabeling
LabelingTechnologies
Technologies
™

IMPORTANT NOTICE: The products described in this manual

in thisaremanual
coveredare
by covered
one or more Limited Use Label License(s). Please refer to thePlease
Appendix onto
802 IMPORTANT NOTICE : The products described by one or more Limited Use Label License(s).
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page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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