Protein Assay by the Bradford Method

Andres, Myka Angelique; Baranda, Jaela Nicole; Buenaflor, Maria Katrina*; Caina, Charisse
Department of Biological Sciences
University of Santo Tomas
España, Manila

Abstract
The individual concentration of a set of protein solutions was determined using the Bradford method. The
absorbance of solutions containing known protein concentrations at 595 nm were plotted against its
respective concentrations, then the plot was used to determine the unknown protein concentration of the
solution. The average concentration of the unknown set is as follows: Unknown 1: 141.5 µg/mL;
Unknown 2: 124.75 µg/mL; Unknown 3: 133.75 µg/mL; Unknown 4: 129.00 µg/mL.

Introduction

Determining the accurate protein concentration is essential for all quantitative measurements of
biochemical interactions. Attributes of a good quantitation assay include the following: rapid,
reliable, and resistant to potentially interfering substances.

In 1976, M. Bradford created a fast and accurate method for quantifying proteins that became the
preferred method in many laboratories. It replaced the classical Lowry method mainly because of
its advantage when it comes to interference with other substances. Bradford method is subject to
less interference with reagents and nonprotein components of the samples, is simpler, faster, and
more sensitive compared to the Lowry method.

The Bradford method depends on the binding of the dye Coomassie Brilliant Blue G-250 to the
protein contained in the sample. Protein concentrations can be estimated by referring to a
standard curve of optical density values of solutions with known protein concentrations.

There are two types of Bradford assay: the standard assay, and the microassay. The standard
assay is appropriate for measuring between 10-100 µg of protein. The microassay on the other
hand, more sensitive than the standard assay, and therefore is useful when the amount of the
unknown protein is limited. It used for detecting between 1-10 µg of protein. Despite its
increased sensitivity, the microassay is more susceptible to interference from other compounds
because addition of greater amounts of sample relative to the dye reagent is required in this form
of assay. In this particular experiment, the standard assay was used.
In this experiment, the group was expected to learn the Bradford method of protein quantitation,
and to accurately determine the protein concentration of the sample assigned to them using the
said method.

Materials and Methods

The materials used in this experiment are protein isolate, Bradford reagent, and Bovine Serum
Albumin Standard. A spectrophotometer was used to measure the absorbance of the protein
solution. A vortex was also utilized to facilitate the mixing of the reagent to the protein solutions.
Each member of the group was given a protein isolate with a concentration that is unique to the
other members of the group.

The Bradford Reagent was prepared by dissolving 100 mg of Coomassie Blue G250 in 50 mL of
95% ethanol. Then, the solution was mixed to 100 mL of 85% phosphoric acid and made up to 1
L by adding distilled water. The reagent was filtered and stored in an amber bottle at room
temperature.

The protein standard used in this experiment was bovine serum albumin. Bovine serum albumin
is a globular protein obtained from cattle. It is frequently used as a standard because it is low-
priced and is easily available in pure form. Also, because many studies have previously used
bovine serum albumin as its protein standard, the results of the assay can be directly compared
with the results in these aforementioned studies. The amount of the protein standard used in this
experiment is 0.20 mg/mL in water.

A set of standards containing 0.20, 0.30, 0.40, 0.50, 0.60, 0.80, and 1.0 mL of bovine serum
albumin stock solution was prepared. A reagent blank consisting of 1.5 mL of distilled water
mixed with 7.5 mL of Bradford reagent was likewise prepared. Distilled water was added to each
tube to bring the volume to 1 mL. 10 mL of Bradford reagent was also added to each tube. 0.10
mL and 0.20 mL of the unknown protein solution was used and was then added with distilled
water to bring its total volume to 1 mL. Using the previously prepared reagent blank, the
spectrophotometer was calibrated to zero. The absorbance of the standards and the unknown
protein solution was read at 595 nm against the reagent blank after 5 minutes but before 1 hour.
The standard curve was made by plotting the concentration of the BSA standards against the
absorbance read (A595). The concentration of the protein solution in reference to the standard
curve for bovine serum albumin was calculated.
 Results and Discussion

Results

The values of the absorbance of the protein standards steadily increases as the concentration of
the protein standards increases, thus, their points were located close to the best fit line.
Therefore, it can be seen that there is a direct relationship between the absorbance of a protein
sample and its protein concentration.

The read A595 of the unknown samples are as follows:

Sample 1 Sample 2 Sample 3 Sample 4

A B A B A B A B
0.293 0.281 0.256 0.251 0.315 0.228 0.270 0.254

Using the information from these two figures, the concentration of each sample was calculated
using the formula y = mx+b. The average concentration of each sample was obtained by adding
the calculated concentrations of A and B for each sample and dividing it by two. Hence, the
computed average concentration for Sample # 1 is 141.5 µg/mL, 124.75 µg/mL for Sample # 2,
133.75 µg/mL for Sample #3, and 129.00 µg/mL for Sample # 4.

Discussion

The Bradford method involves the addition of a cationic (acidic) dye, Coomassie Blue G250, to
the protein-containing solution. Coomassie Blue G250 binds to basic and aromatic amino acids
in proteins. The dye has 4 different ionic forms, its pKa values are 1.15, 1.82, and 12.4. Among
the three charged forms that prevail in the acidic reagent, the more cationic red and green forms
of the dye have a maximum absorbance of 470 nm and 650 nm respectively. However, the more
anionic blue form of the dye, the form that enables the dye to bind itself to proteins, has an
maximum absorbance of 595 nm. Hence, the quantity of protein can be estimated by finding out
the amount of dye in its blue ionic form. This can be made possible by measuring the absorbance
of the solution at 595 nm.

Protein binding to the dye Coomassie Blue G250 could shift the maximum absorbance of the
blue ionic form of the dye to 590-620 nm. While it is recommended that the measurement of a
substance’s absorbance at a higher wavelength, at the normal pH of the assay, a considerable
proportion of the dye is in green form (λmax = 650 nm) interferes with the measurement of
absorbance of the dye-protein mixture at 620 nm. The best option for this is to measure the
absorbance at 595 nm. Measurement at this wavelength maximizes the absorbency because of
the dye-protein mixture and minimizes the absorbency due to the green form of the free dye.

The standard curve is supposed to be nonlinear due to problems induced by the reduction of the
amount of free dye. Measures were taken in order to reduce the incidence of these problems and
to improve the linearity of the assay. Among these measures are the following: plotting the ratio
of absorbance at 595 nm, and measuring the absorbance of each mixture relative to the reagent
blank.
References

Bradford, M. M. (1976). A rapid and senstitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry , 248-
254.

Deutscher, M. P. (1992). Guide to Protein Purification (4th ed., Vol. 182). Gulf Professionall
Publishing.

Rosenberg, I. M. (2005). Protein Analysis and Purification: benchtop techniques. Springer.

Walker, J. M. (1994). Basic Protein and Peptide Protocols. Humana Press.

Walker, J. M. (2002). The Protein Protocols Handbook. Humana Press.