BRADFORD PROTEIN ASSAY
Background & Theory
Four spectroscopic methods are routinely used to determine the concentration of
protein in a solution. These include measurement of the protein's intrinsic UV absorbance
and three methods which generate a protein-dependent color change; the Lowry assay,
the Smith copper/bicinchoninic assay and the Bradford dye assay.
The first, UV absorbance, requires that a pure protein with known extinction
coefficient be used, in a solution free of interfering (UV absorbing) substances. The
Lowry and copper/bicinchoninic assays are based on reduction of Cu2+ to Cu1+ by
amides. Although this makes them potentially quite accurate, they require the
preparation of several reagent solutions, which must be carefully measured and mixed
during the assay. This is followed by lengthy, precisely timed incubations at closely
controlled, elevated temperatures, and then immediate absorbance measurements of the
unstable solutions. Both assays may be affected by other substances frequently present in
biochemical solutions, including detergents, lipids, buffers and reducing agents. This
requires that the assays also include a series of standard solutions, each with a different,
known concentration of protein, but otherwise having the same composition as the
sample solutions.
The Bradford assay is faster, involves fewer mixing steps, does not require heating, and
gives a more stable colorimetric response than the assays described above. Like the other
assays, however, its response is prone to influence from non protein sources, particularly
detergents, and becomes progressively more nonlinear at the high end of its useful protein
concentration range. The response is also protein dependent, and varies with the
composition of the protein. These limitations make protein standard solutions necessary.
Objective
This assay is based on the use of a dye, Coomassie Brilliant Blue G-250, to which protein
binds, altering the light absorbance properties of the dye. When the dye is prepared as an
acidic solution (in 85% phosphoric acid), it maximally absorbs light with a wavelength of
465 nm. Addition of protein results in a shift of the dye's absorption maximum to 595
nm. As the protein concentration increases, the absorbance of light at 595 nm increases
linearly. This increase in absorbance can be measured in a spectrophotometer. Although
the absorbance of Coomassie blue dye at 595 nm is proportional to the amount of protein
bound, it is necessary to establish a correspondence between absorbance values and
known amounts of protein. To do this, you will prepare a series of protein standards –
dilutions of a protein solution of known concentration. Once you have measured the
A595 of each standard, you will be able to plot the A595 as a function of the known
protein content of each standard. After measuring the A595 of unknown sample, the
standard curve then can be used to determine the amount of protein corresponding to the
absorbance values measured.
Protocol
Preparation of bovine serum albumin protein assay standards:
In order to measure and plot a standard curve of protein concentration versus absorbance
at 595 nm, a series of dilutions of the BSA protein standard stock solution must be
prepared. The easiest way to solve for the volume of protein stock solution required for
each dilution is to use the formula C1V1 = C2V2. C1 is the concentration of the protein
stock solution, V1 is the volume of the stock solution required, C2 is the concentration of
the diluted sample, and V2 is the volume of the diluted sample. The concentration of the
stock solution (C1) is 100 µg/ml, the concentration of the diluted sample is (C2), and the
volume of the diluted sample is fixed at 200 µl. Therefore, solving for the volume of
stock solution required: V1 = C2V2/C1
Protein Standards - Protein standards should be prepared in the same buffer as the
samples to be assayed. A convenient standard curve can be made using bovine serum
albumin with concentrations of 0, 10, 20, 30, 40, 50 µg/ml for the microassay (extinction
coefficient of BSA is 0.667).
Bradford Reagent - Bradford reagent can be made by dissolving 100 mg Coomassie
Blue G-250 in 50 ml 95% ethanol, adding 100 ml 85% (w/v) phosphoric acid to this
solution and diluting the mixture to 1 liter with water.
Procedure
1. Prepare a 10-fold dilution of a 1 mg/ml BSA sample by adding 100 µl of 1 mg/ml
BSA to 900 µl of distilled water to make 100µg/ml BSA.
2. Generate test samples for the reference cell, blank, BSA standards and the protein
sample to be tested according to Table 1 in disposable cuvettes.
3. Note that a dilution of the protein sample may be required for the resulting
absorbance to fall within the linear range of the assay.
4. Allow each sample to incubate at room temperature for 5 minutes.
5. Measure the absorbance of each sample at 595 nm using a UV-visible
spectrophotometer. Be sure to allow the instrument to warm up for at least 15 minutes
prior to use.
6. Plot the absorbance of each BSA standard as a function of its theoretical
concentration. The plot should be linear. Determine the best fit of the data to a
straight line in the form of the equation "y = mx + b" where y = absorbance at 595 nm
and x = protein concentration.
7. Use this equation to calculate the concentration of the protein sample based on the
measured absorbance. If the absorbance of the test sample is outside of the
absorbance range for the standards, then the assay must be repeated with a more
appropriate dilution, if any. The linear range for the assay (and for most
spectrophotometers is 0.2 - 0.8 O.D. units).
Table 1: Preparation of test samples for the Bradford protein assay.
Test Sample
Blank
BSA Standard
(10 µg/ml)
BSA Standard
(20 µg/ml)
BSA Standard
(30 µg/ml)
BSA Standard
(40 µg/ml)
BSA Standard
(50 µg/ml)
Protein Sample
Sample vol., Water,
µl
µl
0
200
20
180
Bradford reagent,
µl
800
800
40
160
800
60
140
800
80
120
800
100
100
800
100
100
800