Presentation on theme: "Electro Mobility Shift Assay

EMSA Band Shift Assay Gel Shift Assay"— Presentation

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1 Electro Mobility Shift Assay EMSA Band Shift Assay Gel Shift Assay
R. Voll 09/01

2 Gene Regulation by Transcription Factors

Regulatory RegionCoding SequenceR. Voll 09/01

3 Application: Detection of DNA-binding factors/proteins

Analysis of DNA sequences (e. g. promoter or enhancer regions) for their
potential to bind specifically to proteins/nuclear extractsAnalysis of
(sub-)cellular extracts for the presence of certain DNA-binding proteins (e. g. a
transcription factor with a known recognition sequence)R. Voll 09/01

4 EMSA: Principle Nuclear extract of non-activated cells

Radioaktively labeled oligonucleotidewith NF-B - binding site (probe)and bound
NF-BNF-BRadioactively labeled oligonucleotidewith NF-B - binding site
(probe)Free ProbeA double-stranded oligonucleotide containig a NF-B- binding
site is labeled with a radioactive isotope and incubated with a nuclear
extract.During gel-electrophoresis, NF-B bound to the oligonucleotide causes a
shiftcompared to the free probe.R. Voll 09/01

5 Preparation of Nuclear and Cytosolic Extracts

The procedure is carried out on ice rsp at 4°C and in thepresence of protease
(and phosphatase) inhibitors.1. Swell cells in hypotonic lysis buffer2. Add NP-40
and vortex to disrupt cytoplasmic membrane3. Centrifuge to pellet nuclei4.
Carefully remove supernatant (contains cytosolic andmembrane fraction)4. Wash
nuclear pellet once in lysis buffer5. Add hypertonic extraction buffer to nuclear
pellet6. Agitate vigouresly for 30 minutes7. Centrifuge at high speed8. Remove
nuclear extract, determine protein concentrationand freeze on dry ice until
EMSA is performedR. Voll 09/01

6 The ProbeDouble stranded radiolabeled oligonucleotides containing a

transcription factor binding siteAP-15’-GCT TGA TGA CTC AGC CGG AA C-3’3’-
CGA ACT ACT GAG TCG GCC TT G-5’NF-kB5’-AGT TGA GGG GAC TTT CCC AGG C-
3’3’-TCA ACT CCC CTC AAA GGG TCC G-5’Binding motifR. Voll 09/01

7 Annealing the Oligos Heat up an equimolar mixture of the 2 oligos

to 95°C and let them slowly cool down by turningoff the heat block.R. Voll 09/01
8 Labeling the Probe (I) A. T4 Polynucleotide Kinase
5’-AGT TGA GGG GAC TTT CCC AGG-3’3’-CA ACT CCC CTC AAA GGG TCC G-5’5’-
P-AGT TGA GGG GAC TTT CCC AGG-3’3’-CA ACT CCC CTC AAA GGG TCC G-P-5’+
Adenosin-P-P-P (g-ATP)PNKR. Voll 09/01

9 Labeling the Probe (II)

B. Klenow Fragment of E. coli DNA Polymerase I5’-ACT TGA GGG GAC TTT CCC
AG-3’3’-A ACT CCC CTC AAA GGG TCC G-5’5’-ACT TGA GGG GAC TTT CCC AGG C-
3’3’-TGA ACT CCC CTC AAA GGG TCC G-5’+ a-32-P dGTP + dCTP + dTTPKlenowR.
Voll 09/01

10 Removal of Unincorporated Nucleotides

Remove not incorporated nucleotides bySephadex G50 columnornon-denaturing
PA gel purificationrepeated ethanol precipitationR. Voll 09/01

11 Reagents Competitor DNA: Competition of unspecific

poly (dI-dC) . poly (dI-dC) binding (e. g. histones)BSA: Protection of
nuclearextractsGTP: ?Radiolabeled Probe: Detection of DNA-
bindingproteinsReaction Buffer Binding conditionsR. Voll 09/01

12 Analysis by non-Denaturing Polyacrylamide Gel Electrophoresis

R. Voll 09/01

13 Proof of SpecificitySupershift using antibodies against the DNA-binding

proteinCompetition for binding to the radiolabeled probe usingunlabeled wildtype
and mutated oligosR. Voll 09/01

14 Supershift Nuclear extract of activated cells

cells with anti-p50 antibodySupershiftp50/p65 + anti-p50Radioaktiv labelled
oligonucleotidewith NF-B - binding site (probe)and bound NF-
Bp50/p65Radioactiv labelled oligonucleotidewith NF-B - binding site
(probe)Free probeA double-stranded oligonucleotide containig a NF-B- binding
site is labelled radioactive and incubated with a nuclear extract.During gel-
electrophoresis, NF-B bound to the oligonucleotide causes a shiftcompared to
the free probe.R. Voll 09/01

15 Competition with Unlabeled Oligos

p50/p65p50/p50UnspecificFree probeGGG GAC TTT CCCGGA GAC TTT CCCWild
type oligo Mutated oligoIncreasing amounts of unlabeled oligos containing the
NF-kB binding siteor unlabeled oligos with a mutated binding site were added to
the reaction mixprior to gel electrophoresis. Specific binding is extinguished
only by thenon-mutated oligo.R. Voll 09/01
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