Bio-Rad Protein Assay User Manual
Bio-Rad
Protein Assay
For Technical Service
Call Your Local Bio-Rad Office or
in the U.S. Call 1-800-4BIORAD
(1-800-424-6723)
Table of Contents
Section 1 Introduction..................................................................... 1
1.1 Principle..................................................................................... 1
1.2 Product Description.................................................................... 3
1.3 Materials Required but Not Supplied......................................... 3
Section 2 Instructions...................................................................... 4
2.1 Reconstituting the Standard....................................................... 4
2.2 Standard Procedure.................................................................... 4
2.3 Microassay Procedure................................................................ 5
2.4 Microtiter Plate Protocols .......................................................... 6
Section 3 Common Questions......................................................... 8
Section 4 Troubleshooting Guide ................................................... 12
Section 5 Ordering Information..................................................... 13
Section 6 Safety Information.......................................................... 14
Section 7 References........................................................................ 24
Section 1
Introduction
The Bio-Rad Protein Assay, based on the method of Bradford, is a
simple and accurate procedure for determining concentration of solubilized protein. It involves the addition of an acidic dye to protein solution,
and subsequent measurement at 595 nm with a spectrophotometer or
microplate reader. Comparison to a standard curve provides a relative
measurement of protein concentration.
1.1 Principle
The Bio-Rad Protein Assay is a dye-binding assay in which a differential color change of a dye occurs in response to various concentrations
of protein.1The absorbance maximum for an acidic solution of
Coomassie®Brilliant Blue G-250 dye shifts from 465 nm to 595 nm when
binding to protein occurs.
basic and aromatic amino acid residues, especially arginine.5Spector
found that the extinction coefficient of a dye-albumin complex solution
was constant over a 10-fold concentration range. Thus, Beer’s law may be
applied for accurate quantitation of protein by selecting an appropriate
ratio of dye volume to sample concentration.
Interferences may be caused by chemical-protein and/or chemical-dye
interactions. Table 1 lists those chemical reagents not directly affecting
the development of dye color. (Note: Basic buffer conditions and detergents interfere with this assay.) Since every protein-chemical reagent
combination has not been assayed, it is possible that some of the listed
reagents produce interference in combination with certain proteins.
However, with respect to proteins such as bovine serum albumin and
gamma globulin, the listed reagents show little or no interference. The
acceptable concentrations of reagents for the Standard Procedure are
shown in Table 1. Equivalent concentrations of reagents for the
Microassay Procedure (see Section 2) are 1/40 of those listed in this table,
due to the difference of sample-to-dye ratios between the Standard and
Microassay Procedures.
2,3,4
The Coomassie blue dye binds to primarily
6
1
Table 1. Reagents Compatible with the Bio-Rad Protein Assay
When Using the Standard Procedure.*
Acetate, 0.6 M KCI, 1.0 M
Acetone Malic acid, 0.2 M
Adenosine, 1 mM MgCl
Amino Acids Mercaptoethanol, 1.0 M
Ammonium sulfate, 1.0 M MES, 0.7 M
Ampholytes, 0.5% Methanol
Acid pH MOPS, 0.2 M
ATP, 1 mM NaCl, 5 M
Barbital NAD, 1 mM
BES, 2.5 M NaSCN, 3 M
Boric acid Peptones
Cacodylate-Tris, 0.1 M Phenol, 5%
CDTA, 0.05 M Phosphate, 1.0 M
Citrate, 0.05 M PIPES, 0.5 M
Deoxycholate, 0.1% Polyadenylic acid, 1 mM
Dithiothreitol, 1 M Polypeptides (MW<3000)
DNA, 1 mg/ml Pyrophosphate, 0.2 M
EDTA, 0.1 M rRNA, 0.25 mg/ml
EGTA, 0.05 M tRNA, 0.4 mg/ml
Ethanol total RNA, 0.30 mg/ml
Eagle’s MEM SDS, 0.1%
Earle’s salt solution Sodium phosphate
Formic acid, 1.0 M Streptomycin sulfate, 20%
Fructose Triton X-100, 0.1%
Glucose Tricine
Glutathione Tyrosine, 1 mM
Glycerol, 99% Thymidine, 1 mM
Glycine, 0.1 M Tris, 2.0 M
Guanidine-HCI Urea, 6 M
Hank's salt solution Vitamins
HEPES buffer, 0.1 M
, 1.0 M
2
* Interference may be caused by chemical-protein and/or chemical-dye interactions. Table 1 lists
those chemical reagents not directly affecting the development of dye color. Since every protein-
chemical reagent combination has not been assayed, it is possible that some of the listed reagents
produce interference in combination with certain proteins. However, with respect to proteins such as
bovine albumin and globulin, the above listed reagents show little or no interference.
2
1.2 Product Description
Protein Assay Dye Reagent Concentrate (catalog number 500-0006)
contains 450 ml of solution containing dye, phosphoric acid, and
methanol. One bottle of dye reagent concentrate is sufficient for 450
assays using the standard assay procedure, or 2,250 assays using the
microassay procedure.
The Dye Reagent Concentrate can be purchased in a kit with one of
two standards: Bovine gamma globulin (Kit I, catalog number 500-0001)
or bovine serum albumin (Kit II, catalog number 500-0002).
The Bio-Rad Protein Assay is for research use only.
1.3 Materials Required but Not Supplied
For standard assay
Spectrophotometer set to 595 nm
Cuvettes with 1 cm path length matched to laboratory spectropho-
tometer. Bio-Rad’s disposable polystyrene cuvettes (catalog number
223-9950) are recommended
13 x 100 mm test tubes
Test tube rack for 13 x 100 mm test tubes
Vortex mixer
Whatman #1 filter (or equivalent) and funnel for dye reagent prepara-
tion
Graduated cylinders, pipets, and containers for reagent preparation
and storage
Pipets accurately delivering 100 µl and 5.0 ml
For microplate assay
Microtiter plates
Pipets accurately delivering 200 µl and 800 µl
3
Section 2
Instructions
2.1 Reconstituting the Standard
To reconstitute the lyophilized bovine gamma globulin and bovine
serum abumin standards, add 20 ml of deionized water and mix until dissolved. If the standard will not be used within 60 days, it should be
aliquoted and frozen at -20 °C.
Note: The standards contain buffer salts required for solubilizing the protein.
2.2 Standard Procedure
1. Prepare dye reagent by diluting 1 part Dye Reagent Concentrate with
4 parts distilled, deionized (DDI) water. Filter through Whatman #1
filter (or equivalent) to remove particulates. This diluted reagent may
be used for approximately 2 weeks when kept at room temperature.
2. Prepare three to five dilutions of a protein standard, which is repre-
sentative of the protein solution to be tested. The linear range of the
assay for BSA is 0.2 to 0.9 mg/ml, whereas with IgG the linear range
is 0.2 to 1.5 mg/ml. (See Common Questions, question 4, for more
information.)
3. Pipet 100 µl of each standard and sample solution into a clean, dry
test tube. Protein solutions are normally assayed in duplicate or tripli-
cate.
4. Add 5.0 ml of diluted dye reagent to each tube and vortex.
5. Incubate at room temperature for at least 5 minutes. Absorbance will
increase over time; samples should incubate at room temperature for
no more than 1 hour.
6. Measure absorbance at 595 nm.
4
Standard procedure
(20-140 µg)
Protein (mg/ml)
BSA
IgG
O.D.
595
0.20
0.2
0.4
0.6
0.40 0.60 0.80 1.00 1.20 1.40
0.8
1.0
Fig. 1. Typical standard curve for the Bio-Rad Protein Assay, bovine gamma
globulin (standard I), bovine serum albumin (standard II). O.D.
blank - 200-1,400 µg/ml x 0.1 ml = 20-140 µg protein.
corrected for
595
2.3 Microassay Procedure
1. Prepare three to five dilutions of a protein standard which is represen-
tative of the protein solution to be tested. The linear range of the assay
for BSA is 1.2 to 10.0 µg/ml, whereas with IgG the linear range is 1.2
to 25 µg/ml. (See Common Questions, question 4, for more informa-
tion.)
2. Pipet 800 µl of each standard and sample solution into a clean, dry
test tube. Protein solutions are normally assayed in duplicate or tripli-
cate.
3. Add 200 µl of dye reagent concentrate to each tube and vortex.
4. Incubate at room temperature for at least 5 minutes. Absorbance will
increase over time; samples should incubate at room temperature for
no more than 1 hour.
5. Measure absorbance at 595 nm.
5
Fig. 2. Typical standard curve for the Bio-Rad Protein Microassay (1-20 µg/ml),
Microassay procedure
(1-20 µg)
Protein (µg,ml)
BSA
IgG
O.D.
595
2.5 5 7.5
10
12.5 15 17.5 20
22.5
25
0.1
0.2
0.3
0.4
0.5
0.6
bovine gamma globulin (standard I), bovine serum albumin (standard II).
O.D.
corrected for blank. 1.25-25 µg/ml x 0.8 ml = 1-20 µg protein.
595
2.4 Microtiter Plate Protocols
The Bio-Rad Protein Assay can also be used with a microplate reader,
such as Bio-Rad's Model 450 and 3550 Microplate Readers. The linear
range of the Standard and Microassay procedures when used in the
microtiter plate format is slightly changed, since the ratio of sample to dye
is modified.
Standard Procedure for Microtiter Plates
1. Prepare dye reagent by diluting 1 part Dye Reagent Concentrate with
2. Prepare three to five dilutions of a protein standard, which is repre-
4 parts DDI water. Filter through a Whatman #1 filter (or equivalent)
to remove particulates. This diluted reagent may be used for about 2
weeks when kept at room temperature.
sentative of the protein solution to be tested. The linear range of this
microtiter plate assay is 0.05 mg/ml to approximately 0.5 mg/ml.
Protein solutions are normally assayed in duplicate or triplicate.
6
3. Pipet 10 µl of each standard and sample solution into separate
microtiter plate wells.
4. Add 200 µl of diluted dye reagent to each well. Mix the sample and
reagent thoroughly using a microplate mixer. Alternatively, use a
multi-channel pipet to dispense the reagent. Depress the plunger
repeatedly to mix the sample and reagent in the well. Replace with
clean tips and add reagent to the next set of wells.
5. Incubate at room temperature for at least 5 minutes. Absorbance will
increase over time; samples should incubate at room temperature for
no more than 1 hour.
6. Measure absorbance at 595 nm.
Microassay Procedure for Microtiter Plates
1. Prepare three to five dilutions of a protein standard, which is repre-
sentative of the protein solution to be tested. The linear range of the
assay is 8.0 µg/ml to approximately 80 µg/ml. Protein solutions are
normally assayed in duplicate or triplicate.
2. Pipet 160 µl of each standard and sample solution into separate
microtiter plate wells.
3. Add 40 µl of dye reagent concentrate to each well. Mix the sample
and reagent thoroughly using a microplate mixer. Alternatively, use a
multi-channel pipet to dispense the reagent. Depress the plunger
repeatedly to mix the sample and reagent in the well. Replace with
clean tips and add reagent to the next set of wells.
4. Incubate at room temperature for at least 5 minutes. Absorbance will
increase over time; samples should incubate at room temperature for
no more than 1 hour.
5. Measure absorbance at 595 nm.
7
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