Standard Curve Preparation for
Determining Protein Content

concentration of the substance and the path
length of the light through the solution.

Cell Fractionation and Separation

Protein Quantitation — isolation,
characterization, purification, and

Homogenization, to break open cells to
separate their structural and molecular

Procedures that need qunatified protein
• chromatography
• electrophoresis
• functional assays
• immunochemical separation &

Homogenization Methods
• Detergents (SDS)
• Salts for Osmotic Alteration
• Enzymes (trypsin and proteinase K)
• Mechanical methods
• Ultrasonification (sound waves)

280 nm, abs of protein molecules in solutions
due to presence of aromatic amino acids
(tyr & trp)
Zero Buffer Absorbance, to resolve
interference of substance in the buffer for

Fractionation, separation or subcellular
organelles by centrifugation (by density)
Pellet — material that collects at bottom of
microfuge tube
Supernatant — fluid above the pellet

• Crude, homogenized material, all
Colorimetric Methods of Quantitation
• Protein dye-binding Chemistry
• BCA™ Protein Assay
• Modified Lowry Protein Assay
• Protein-copper Chelation Chemistry
Selection of Protein Standard
• h i g h l y p u r i fi e d v e r s i o n o f m o s t
predominant protein in sample
• Bovine Serum Albumin (BSA)
Biuret Assay
• Biuret, small compound formed when
urea is heated causing two urea
molecules to join. Copper complexes
produced produce strong blue color.
Bradford Assay
• Coomassie Brilliant Blue G-250
—binds to basic (especially Arg) and
aromatic amino acid residues
• Cationic (Red) [470 nm]
• Neutral (Green)
• Anionic (Blue) [595 nm]
Beer’s Law, the quantity of light absorbed by
a substance dissolved in a fully transmitting
solvent is directly proportional to the

components present
• Nuclei (pellet)
• Soluble (supernatant)
• Microsome (pellet)
595 nm, abs to be read at

Marker Enzyme Assay
Marker Enzymes — enzymes localized in a
particular organelle of the cell
A. Alkaline Phosphodiesterase (APDE)
• present in phagocytic vacuoles
• thymidine 5’ monoP —> p-nitrophenol
• yellow compound

• Buffer D (Tris-borate, Triton X-100,
MgCl2, ZnSO4)

• Substrate D (p-nitrophenyl thymidine 5’

‘chain breaker’ because of its cyclic structure glycine. Acid Phosphatase Assay • Present in Lysosomes • pNPP or pNTP —> para-nitrophenol • Yellow Compound • Buffer C (glycine-HCl. responsible for damaging DNA SYBR Green — dramatic fluorescence in presence of double stranded DNA SDS-Polyacrylamide Gel Electrophoresis Gel Electrophoresis — separates charged molecules by running through a matrix (gel) in an electrical current. carboxylate group. mabilis magfold lol Primary Structure .g proline. the exact amino acid sequence can be deciphered and thus will show more the inherent properties of the protein being investigated. measure of fluorescence • BSA or Chicken Egg White Albumin • Flamingo™ Pink Fluorescent Stain DNAse — key enzymes released during apoptosis. products bind to Flamingo™ —> enhanced fluorescence • Relative Fluorescence Units (RFU). Amino Acid = Residue Amino Acid – alpha carbon. folding . R group (gives property) Affects Structure of the Protein e.B. Peroxidase Assay • present in peroxisomes • Peroxidase oxidized reduced TMB in presence of H2O2 • Blue color SDS .Sodium Dodecyl Sulfate • gives proteins net negative charge • removes 2ndary and 3rtiary structures Ammonium Persulfate and TEMED “POISONOUS” • Tetramethyl Benzidine (TMB) C. may connote some physiological conditions such as stress • Differential Scanning Fluorimetry (DSF) • Proteases digest BSA. concentrate and properly align protein samples • 12% Acrylamide — Running Gel. Triton X-100) • Substrate C (p-nitrophenol phosphate) D. separates individual polypeptides into discrete bands Staining • Coomassie Blue R250 • SYPRO Ruby SDS-PAGE Gel Image Analysis Protein Profiling — data from SDS-PAGE used to determine how closely related two or more species are at the level of expressed gene products Protein Sequence Analysis and Homology Modeling of 3D Structures Reverse Genetics — Once the gene sequence is known. Nucleic Acids — net negative charge due to their phosphate group Cathode (NEGATIVE). amino group. Anode (POSITIVE) Mini-Protean III — vertical slab unit designed for faster electrophoresis • 4% Acrylamide — Stacking Gel.amino acid sequence Secondary Structure – structure. Mitochondrial Reductase/ Dehydrogenase Assay • Reduction of Blue Resazurin —> Red Resofurin • Alamar Blue® Viability Reagent E. Protease Acitvity Assay • Protease. restricts protein migration.

Plots phi and psi angles. still a type of secondary structure 4.Structures are conserved within the family of the proteins evolution Ramachandran Plot .Types of Secondary Structures 1. Turn – not necessarily for the alpha helix Tertiary Structure – 3d Quaternary Structure .Refers to the process wherein the structure of the protein is predicted based on a template . Random coil – randomly coiled. a crystal structure (?) . Xray Crystallography/Protein Crystallography – determining 3d protein structure **you need to have a protein signal Crystal – ordered periodic material **more reliable than NMR NMR – Nuclear Magnetic Resonance Concept: Poly exclusion principle Spots correspond to the structure of the protein Downside: difficult to interpret *like taking a video of the protein. corresponds to the tortional angles of the peptides o Alpha carbon and the nitrogen Tortional angle – between alpha carbon and nitrogen Psi angle – between carbonyl carbon and alpha carbon Omega Angle – not included in the ramchandran plot ♣ Between carbonyl carbon and nitrogen amide bond ♣ It does not give you any idea about the protein structure ♣ Becausde of its rigidity (partial double bond character). measuring the structure in solution.4 amino acid residue per turn. beta sheath 3. Maintained by Hydrogen bonding 2.alpha helix – glycine on turn position. trimeric. Why bother? Structure-function relationship Homology Modelling . multimeric.6/3.The template must be well-known. 3.subunits Dimeric. etc. hindi masyadong nakakaikot .