in the name of god

In the name of

God

Summer School

Influenza Unit,Pasteur Institute of Iran

summer 2012

PROTEINS

Assay Methods(Protein

quantitation)B.Farahmand

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INTRODUCTION

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

Amino acids

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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

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پروتئینها فیزیکوشیمیایی خصوصیات

شکل •اندازه•بارالکتریکی•

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Protein Estimation is a part of any laboratory workflow

involving protein extraction, purification, labeling and

analysis.

METHODS OF PROTEIN ESTIMATION

Biuret method Folin- Lowry method Bradford method Bicinchoninic method UV method Flourimetric method Kjeldahl method Mass Spectrometry

Colorimetrc assay

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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

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BIURET TESTBIURET TEST

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

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

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Folin-Ciocalteu ( Lowry ) Assay

Step 1

Step 2

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Comparison of Lowry and Biuret

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Bicinchoninic method

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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

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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).

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Mechanism of Dye response and interference in the Bradford protein assay

Anionic dye

Protonated or cationic amino acids

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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

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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.

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Selecting a Protein Assay & a Standard

protein

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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

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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.

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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).

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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

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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.

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Methods

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Biosafety in protein assays

• Wear Gloves and Labcoat

• MSDS (Material Safety Data Sheet) Folin reagent, Phosphoric acid, ……

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Standard Curve

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Standard Curve preparation

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A750nm

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.

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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

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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.

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Instrument for Lowery assay

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Instrument for Bradford assay

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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.

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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.

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Indirect calculation

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Indirect calculation

• C= Concentration• OD= Optical Density• tgα=Slope of standard curve• tgα=∆Cs/∆ODs

• CX = tgα × ODX

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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).

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Thanks