1. In the name of God

2. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

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

4. INTRODUCTION Proteins are highly complex natural compounds composed of large number of different amino acids.

5. Amino acids Summer School

7. 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

9. خصوصیات فیزیکوشیمیایی پروتئینها شکل اندازه بارالکتریکی Summer School

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

11. 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

12. 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

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

14. 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

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

16. Comparison of Lowry and Biuret Summer School

17. Bicinchoninic method Summer School

18. 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

19. 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

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

21. 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

22. Comparison of standard curve of Bradford, Lowry and BCA assay 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

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

24. 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

25. 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

26. 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

27. 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

28. 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

29. Methods Summer School

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

31. Standard Curve Summer School

32. Standard Curve preparation Summer School A750nm

33. Comments for standard preparation For greatest accuracy in estimating total protein concentration in unknown samples, it is essential to include a standard curve each time the assay is performed. This is particularly true for the protein assay methods that produce non- linear standard curves. Deciding on the number of standards and replicates used to define the standard curve depends upon the degree of non-linearity in the standard curve and the degree of accuracy required. In general, fewer points are needed to construct a standard curve if the color response is linear. Typically, standard curves are constructed using at least two replicates for each point on the curve. Summer School

34. Sample Preparation for Protein Assays it must be solubilized inhibit microbial growth avoid casual contamination of the sample by foreign debris such as dust, hair, skin or body oils. After filtration or centrifugation to remove the cellular debris, typical samples will still include nucleic acids, lipids and other non-protein compounds. nonprotein components (detergents, biocides or antimicrobial agents, protease inhibitors, different salts, denaturants, reducing agents and chaotropes) are critical for choosing an appropriate assay Summer School

35. Strategies for interfering substance elimination Choose a different protein assay method or a version of the same assay method that includes components to overcome the interference. Dialyze or desalt the sample to remove interfering substances that are small (i.e., less than 1000 daltons), such as reducing agents. Precipitate the protein in TCA or other appropriate reagent, remove the solution containing the interfering component, and then redissolve the protein for analysis. Summer School

36. Instrument for Lowery assay Summer School

37. Instrument for Bradford assay Summer School

38. 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

39. 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

40. Indirect calculation Summer School

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

42. Comments Obviously, the most accurate results are possible only when unknown and standard samples are treated identically. This includes assaying them at the same time and in the same buffer conditions, if possible. Because different pipetting steps are involved, replicates are necessary if one wishes to calculate statistics (e.g., standard deviation, coefficient of variation) to account for random error. Although most modern spectrophotometers and plate readers have built- in software programs for protein assay data analysis, several factors are frequently misunderstood by technicians. Taking a few minutes to study and correctly apply the principles involved in these calculations can greatly enhance one's ability to design assays that yield the most accurate results possible (see the related Tech Tips and links). Summer School

43. Thanks