Download - In the name of God
![Page 1: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/1.jpg)
In the name of
God
![Page 2: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/2.jpg)
Summer School
Influenza Unit,Pasteur Institute of Iran
summer 2012
![Page 3: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/3.jpg)
PROTEINS
Assay Methods(Protein
quantitation)B.Farahmand
Summer School
![Page 4: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/4.jpg)
INTRODUCTION
• Proteins are highly complex natural compounds composed of large number of different amino acids.
![Page 5: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/5.jpg)
Amino acids
Summer School
![Page 6: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/6.jpg)
Summer School
![Page 7: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/7.jpg)
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
![Page 8: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/8.jpg)
Summer School
![Page 9: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/9.jpg)
پروتئینها فیزیکوشیمیایی خصوصیات
شکل •اندازه•بارالکتریکی•
Summer School
![Page 10: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/10.jpg)
Protein Estimation is a part of any laboratory workflow
involving protein extraction, purification, labeling and
analysis.
![Page 11: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/11.jpg)
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
![Page 12: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/12.jpg)
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
![Page 13: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/13.jpg)
BIURET TESTBIURET TEST
By reducing the copper ion from cupric to cuprous form, the reaction produces a faint blue-violet color Summer School
![Page 14: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/14.jpg)
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
![Page 15: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/15.jpg)
Folin-Ciocalteu ( Lowry ) Assay
Step 1
Step 2
Summer School
![Page 16: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/16.jpg)
Comparison of Lowry and Biuret
Summer School
![Page 17: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/17.jpg)
Bicinchoninic method
Summer School
![Page 18: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/18.jpg)
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
![Page 19: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/19.jpg)
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
![Page 20: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/20.jpg)
Mechanism of Dye response and interference in the Bradford protein assay
Anionic dye
Protonated or cationic amino acids
Summer School
![Page 21: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/21.jpg)
Dye-Binding ( Bradford ) Assay• Adventage• Fast and inexpensive • Highly specific for protein • Very sensitive [1-20 µg (micro assay) 20-200 µg (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
![Page 22: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/22.jpg)
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
![Page 23: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/23.jpg)
Selecting a Protein Assay & a Standard
protein
Summer School
![Page 24: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/24.jpg)
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
![Page 25: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/25.jpg)
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
![Page 26: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/26.jpg)
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
![Page 27: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/27.jpg)
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.
BCA(Note 1)
MicroBCA ModifiedLowry CoomassiePlusCoomassie(Bradford)
Pierce660 nm
1. Albumin, bovine serum 1.00 1.00 1.00 1.00 1.00 1.00
2. Aldolase, rabbit muscle 0.85 0.80 0.94 0.74 0.76 0.83
3. -Chymotrypsinogen 1.14 0.99 1.17 0.52 0.48 —
4. Cytochrome C, horse heart 0.83 1.11 0.94 1.03 1.07 1.22
5. Gamma Globulin, bovine 1.11 0.95 1.14 0.58 0.56 0.51
6. IgG, bovine 1.21 1.12 1.29 0.63 0.58 —
7. IgG, human 1.09 1.03 1.13 0.66 0.63 0.57
8. IgG, mouse 1.18 1.23 1.20 0.62 0.59 0.48
9. IgG, rabbit 1.12 1.12 1.19 0.43 0.37 0.38
10. IgG, sheep 1.17 1.14 1.28 0.57 0.53 —
11. Insulin, bovine pancreas 1.08 1.22 1.12 0.67 0.60 0.81
12. Myoglobin, horse heart 0.74 0.92 0.90 1.15 1.19 1.18
13. Ovalbumin 0.93 1.08 1.02 0.68 0.32 0.54
14. Transferrin, human 0.89 0.98 0.92 0.90 0.84 0.8
15. a-Lactalbumin — — — — — 0.82
16. Lysozyme — — — — — 0.79
17. Trypsin inhibitor, soybean — — — — — 0.38
Average ratio 1.02 1.05 1.09 0.73 0.68 0.74
Standard Deviation 0.15 0.12 0.13 0.21 0.26 0.27
Coefficient of Variation 14.7% 11.4% 11.9% 28.8% 38.2% 37%
Relative Uniformity High High High Medium Low (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
![Page 28: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/28.jpg)
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
![Page 29: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/29.jpg)
Methods
Summer School
![Page 30: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/30.jpg)
Biosafety in protein assays
• Wear Gloves and Labcoat
• MSDS (Material Safety Data Sheet) Folin reagent, Phosphoric acid, ……
Summer School
![Page 31: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/31.jpg)
Standard Curve
Summer School
![Page 32: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/32.jpg)
Standard Curve preparation
Summer School
A750nm
![Page 33: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/33.jpg)
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
![Page 34: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/34.jpg)
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
![Page 35: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/35.jpg)
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
![Page 36: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/36.jpg)
Instrument for Lowery assay
Summer School
![Page 37: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/37.jpg)
Instrument for Bradford assay
Summer School
![Page 38: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/38.jpg)
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
![Page 39: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/39.jpg)
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
![Page 40: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/40.jpg)
Indirect calculation
Summer School
![Page 41: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/41.jpg)
Indirect calculation
• C= Concentration• OD= Optical Density• tgα=Slope of standard curve• tgα=∆Cs/∆ODs
• CX = tgα × ODX
Summer School
![Page 42: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/42.jpg)
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
![Page 43: In the name of God](https://reader034.vdocuments.us/reader034/viewer/2022051620/56812f7f550346895d950114/html5/thumbnails/43.jpg)
Thanks