In the name of God

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Influenza Unit, Pasteur Institute of Iran summer 2013

PROTEINS Assay Methods (Protein quantitation)

B.Farahmand

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INTRODUCTION

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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

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

Proteins

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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 Summer School

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

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

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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

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

BIURET 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 Summer School

Folin-Ciocalteu ( Lowry ) Assay

Step 1 Step 2

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Comparison of Lowry and Biuret

Lowry reaction

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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 Summer School

Dye-Binding ( Bradford ) Assay

• • • • 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).

• • Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990). 248-254. 1976. Summer School

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

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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

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.

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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 Summer School

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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Standard Protein Selection

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.

1. Albumin, bovine serum 2. Aldolase, rabbit muscle 3. a -Chymotrypsinogen 4. Cytochrome C, horse heart 5. Gamma Globulin, bovine 6. IgG, bovine 7. IgG, human 8. IgG, mouse 9. IgG, rabbit 10. IgG, sheep 11. Insulin, bovine pancreas 12. Myoglobin, horse heart 13. Ovalbumin 14. Transferrin, human 15. a-Lactalbumin 16. Lysozyme 17. Trypsin inhibitor, soybean

Average ratio Standard Deviation Coefficient of Variation Relative Uniformity BCA

(Note 1) 1.00

0.85

1.14

0.83

1.11

1.21

1.09

1.18

1.12

1.17 1.08

0.74

0.93

0.89

— — —

1.02

0.15

14.7% High Micro BCA

1.00

0.80

0.99

1.11

0.95

1.12

1.03

1.23

1.12

1.14

1.22

0.92

1.08

0.98

— — —

1.05

0.12

11.4% High Modified Lowry

1.00

0.94

1.17

0.94

1.14

1.29

1.13

1.20

1.19

1.28

1.12

0.90

1.02

0.92

— — —

1.09

0.13

11.9% High Coomassie Plus

1.00

0.74

0.52

1.03

0.58

0.63

0.66

0.62

0.43

0.57

0.67

1.15

0.68

0.90

— — —

0.73

0.21

28.8% Medium Coomassie (Bradford)

1.00

0.76

0.48

1.07

0.56

0.58

0.63

0.59

0.37

0.53

0.60

1.19

0.32

0.84

— — —

0.68

0.26

38.2% Low

(Note 2)

Pierce 660 nm

0.54

0.8

0.82

0.79

0.38

0.74

0.27

37% Low

1.00

0.83

— 1.22

0.51

— 0.57

0.48

0.38

— 0.81

1.18

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 Summer School

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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Biosafety in protein assays

• Wear Gloves and Labcoat • MSDS (Material Safety Data Sheet) Folin reagent, Phosphoric acid, … … Summer School

Practical work

• Bradford assay • Lowry assay

Steps of assays

• • • • • Standard solution preparation Absorbance or Optical Density reading of Standards Standard curve drawing tgα calculation Unknown sample estimation Summer School

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 . Summer School

Unknown sample concentration calculation

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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

Standard Curve

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Indirect calculation

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Indirect calculation

• • • • • C= Concentration OD= Optical Density tgα=Slope of standard curve tgα=∆C s /∆OD s C X = tgα × OD X Summer School

Thanks