Transcript Slide 1

In the name of
God
Summer School
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
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Proteins
• Proteins are highly
complex natural
compounds composed
of large number of
different amino acids.
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Amino acids
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Levels of Protein Organization
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Primary structure = linear chain of
amino acids
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• Secondary structure = domains of
repeating structures, such as β-pleated
sheets and α-helices
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• Tertiary structure = 3-dimensional
shape of a folded polypeptide, maintained
by disulfide bonds, electrostatic
interactions, hydrophobic effects
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• Quaternary structure = several
polypeptide chains associated together to
form a functional protein
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Physico-chemical properties of proteins
• Shape
• Size
• Electrical charge
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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
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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 TEST
By reducing the copper ion from cupric to cuprous form, the reaction
produces a faint blue-violet color
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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
Lowry reaction
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Bicinchoninic method
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BCA Test
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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
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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: 248-254. 1976.
Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).
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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
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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 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:
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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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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.
BCA
(Note 1)
Micro
BCA
Modified
Lowry
Coomassie
Plus
Coomassie
(Bradford)
Pierce
660 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. a-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
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—
—
—
—
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
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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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Biosafety in protein assays
•
Wear Gloves and Labcoat
•
MSDS (Material Safety Data Sheet)
Folin reagent, Phosphoric acid, …
…
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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
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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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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=∆Cs/∆ODs
• CX = tgα × ODX
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Thanks