1. In the name of God

2. Summer School Influenza Unit, Pasteur Institute of Iran summer 2013

3. PROTEINS Assay Methods (Protein quantitation) B.Farahmand Summer School

4. INTRODUCTION Summer School

5. Protein quantitation is often necessary prior to handling protein samples for isolation and characterization is a required step before chromatographic, electrophoretic and immunochemical analyses Summer School

6. Proteins Proteins are highly complex natural compounds composed of large number of different amino acids. Summer School

7. Amino acids Summer School

8. Levels of Protein Organization Primary structure = linear chain of amino acids Secondary structure = domains of repeating structures, such as β-pleated sheets and α-helices Tertiary structure = 3-dimensional shape of a folded polypeptide, maintained by disulfide bonds, electrostatic interactions, hydrophobic effects Quaternary structure = several polypeptide chains associated together to form a functional protein Summer School

10. Physico-chemical properties of proteins Shape Size Electrical charge Summer School

11. Protein Estimation is a part of any laboratory workflow involving protein extraction, purification, labeling and analysis. Summer School

12. METHODS OF PROTEIN ESTIMATION  Biuret method  Folin- Lowry method  Bradford method  Bicinchoninic method  UV method  Flourimetric method  Kjeldahl method  Mass Spectrometry Colorimetrc assay Summer School

13. Chemistry of Protein Assays Copper-based Protein Assays: – Biuret Protein Assays – Lowry Assay – BCA Protein-copper chelation and secondary detection of the reduced copper Dye-based Protein Assays: – Coomassie (Bradford) Assay Protein-dye binding and direct detection of the color change associated with the bound dye Summer School

14. BIURET TEST By reducing the copper ion from cupric to cuprous form, the reaction produces a faint blue-violet color Summer School

15. Biuret Test Adventage Reproduciple Very few interfering agents (ammonium salts being one such agent ) Fewer deviations than with the Lowry or ultraviolet absorption methods Disadventage Requires large amounts protein (1-20mg) Low sensitivity Summer School

16. Folin-Ciocalteu ( Lowry ) Assay Step 1 Step 2 Summer School

17. Comparison of Lowry and Biuret Summer School Lowry reaction

18. Bicinchoninic method Summer School

19. BCA Test Adventage The color complex is stable There is less suceptibility to detergents Fewer deviations than with the Lowry or Beradford methods Disadventage Bicinchonic acid is expensive Summer School

20. Dye-Binding ( Bradford ) Assay Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976. Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990). CBBG primarily responds to arginine residues (eight times as much as the other listed residues) If you have an arginine rich protein, You may need to find a standard that is arginine rich as well. CBBG binds to these residues in the anionic form Absorbance maximum at 595 nm (blue) The free dye in solution is in the cationic form, Absorbance maximum at 470 nm (red). Summer School

21. Mechanism of Dye response and interference in the Bradford protein assay Anionic dye Protonated or cationic amino acids Summer School

23. Dye-Binding ( Bradford ) Assay Adventage Fast and inexpensive Highly specific for protein Very sensitive [ 1-20 µ g (micro assay) 20-200 µ g (macro assay)] Compatible with a wide range of substances Extinction co-efficient for the dye-protein complex is stable over 10 orders of magnitude (assessed in albumin) Dye reagent complex is stable for approximately one hour Disadventage Non-linear standard curve over wide ranges Response to different proteins can vary widely, choice of standard is very important Summer School

24. Comparison of standard curve of Bradford, Lowry and BCA assays Absorption spectra of anionic and cationic forms of the dye overlap. So the standard curve is non-linear. The assay performs linearly over short concentration stretches. Summer School

25. Selecting a Protein Assay & a Standard protein Summer School

26. Important criteria for choosing an assay include: Compatibility with the sample type and components Assay range and required sample volume Protein-to-protein variation Speed and convenience for the number of samples to be tested Availability of spectrophotometer or plate reader necessary to measure the color produced (absorbance) by the assay Summer School

27. Selecting a Protein Standard If a highly purified version of the protein of interest is not available or it is too expensive to use as the standard, the alternative is to choose a protein that will produce a very similar color response curve in the selected protein assay method and is readily available to any laboratory at any time. Summer School

28. Examples of Standard Protein Generally, bovine serum albumin (BSA) works well for a protein standard because it is widely available in high purity and relatively inexpensive. Alternatively, bovine gamma globulin (BGG) is a good standard when determining the concentration of antibodies because BGG produces a color response curve that is very similar to that of immunoglobulin G (IgG). Summer School

29. Protein-to-protein variation of Thermo Scientific Pierce Protein Assays. For each of the protein assays presented here, 14 proteins were assayed using the standard test tube protocol. The net (blank corrected) average absorbance for each protein was calculated. The net absorbance for each protein is expressed as a ratio to the net absorbance for BSA (e.g., a ratio of 0.80 means that the protein produces 80% of the color obtained for an equivalent mass of BSA). All protein concentrations were at 1000µg/mL, except for those used in the Micro BCA Assay which were at a concentration of 10µg/mL.Thermo Scientific Pierce Protein Assays BCA BCA (Note 1) Micro BCA Modified Lowry Coomassie Plus Coomassie (Bradford) Pierce 660 nm 1. Albumin, bovine serum1.00 2. Aldolase, rabbit muscle0.850.800.940.740.760.83 3.  -Chymotrypsinogen 1.140.991.170.520.48— 4. Cytochrome C, horse heart0.831.110.941.031.071.22 5. Gamma Globulin, bovine1.110.951.140.580.560.51 6. IgG, bovine1.211.121.290.630.58— 7. IgG, human1.091.031.130.660.630.57 8. IgG, mouse1.181.231.200.620.590.48 9. IgG, rabbit1.12 1.190.430.370.38 10. IgG, sheep1.171.141.280.570.53— 11. Insulin, bovine pancreas1.081.221.120.670.600.81 12. Myoglobin, horse heart0.740.920.901.151.191.18 13. Ovalbumin0.931.081.020.680.320.54 14. Transferrin, human0.890.980.920.900.840.8 15. a-Lactalbumin—————0.82 16. Lysozyme—————0.79 17. Trypsin inhibitor, soybean—————0.38 Average ratio1.021.051.090.730.680.74 Standard Deviation0.150.120.130.210.260.27 Coefficient of Variation14.7%11.4%11.9%28.8%38.2%37% Relative UniformityHigh MediumLow (Note 2)Low Notes: 1. The BCA - Reducing Agent Compatible (BCA-RAC) Assay also produced a low coefficient of variation. 2. The Bio-Rad Bradford Protein Assay tested with the same proteins as our Coomassie (Bradford) Assay produced a very high coefficient of variation (46%), corresponding to very low relative uniformity Standard Protein Selection Summer School

30. Protein-to-Protein Variation Each protein in a sample responds uniquely in a given protein assay. Such protein-to-protein variation refers to differences in the amount of color (absorbance) obtained when the same mass of various proteins is assayed concurrently by the same method. These differences in color response relate to differences in: - amino acid sequence, - isoelectric point (pI), - secondary structure - and the presence of certain side chains or prosthetic groups. Depending on the sample type and purpose for performing an assay, protein-to-protein variation is an important consideration in selecting a protein assay method and in selecting an appropriate assay standard (e.g., BSA vs. BGG). Protein assay methods based on similar chemistry have similar protein-to-protein variation. Summer School

31. Biosafety in protein assays Wear Gloves and Labcoat MSDS (Material Safety Data Sheet) Folin reagent, Phosphoric acid, … … Summer School

32. Practical work Bradford assay Lowry assay

33. Steps of assays Standard solution preparation Absorbance or Optical Density reading of Standards Standard curve drawing tgα calculation Unknown sample estimation Summer School

34. Instrument for Lowery assay Summer School

35. Instrument for Bradford assay Summer School

36. Calculations and Data Analysis Note: With most protein assays, sample protein concentrations are determined by comparing their assay responses to that of a dilution-series of standards whose concentrations are known. Protein samples and standards are processed in the same manner by mixing them with assay reagent and using a spectrophotometer to measure the absorbances. The responses of the standards are used to plot or calculate a standard curve. Absorbance values of unknown samples are then interpolated onto the plot or formula for the standard curve to determine their concentrations. Summer School

37. Unknown sample concentration calculation Direct calculation Absorbance values of unknown samples are then interpolated onto the plot Indirect calculation formula for the standard curve to determine their concentrations. Summer School

38. Standard Curve Summer School

39. Indirect calculation Summer School

40. Indirect calculation C= Concentration OD= Optical Density tgα=Slope of standard curve tgα=∆C s /∆OD s C X = tgα × OD X Summer School

41. Thanks