Bradford & BCA Assays

By Grant Akalonu
Instructor: Binh Ngyuen
CHE 447-01
5/28/17
Grant Akalonu Bradford & BSA

Abstract:

In this experiment, the concentration of the protein Bovine Serum Albumin (BSA) was

measured quantitatively using Bradford assay and BCA assay. The concentrations were measured

by taking advantage of the direct relationship between absorbance and concentration, as

demonstrated in Beers Law. The extinction coefficient was determined by plotting graphs of the

absorbance and concentration. For the Bradford assay, the extinction coefficient was determined

to be 0.0472 with an R2 value of 0.961. For the BSA, the extinction coefficient was determined to

be 0.0037 with an R2 value of 0.996.

Introduction

The Bradford assay, is a colorimetric method of determining the concentration of amino a

protein in solution. The assay uses a dye known as Coommasie Brilliant Blue G-250, which

induces a color change in solution in response to a change in the concentration of protein. The

structure of the dye is shown in Figure 1. The dye binds primarily to aromatic and basic amino

acid residues, and changes the maximum absorbance of the dye changes from 465 nm to 595 nm

when it is bounded to protein [1]. In acidic conditions, the red form of the dye will change to the

blue form and bind to the protein of interest [2]. The dye then forms a strong noncovalent

interaction with the protein, via Van der Waals and electrostatic interactions [2]. During the

formation of the protein-dye complex, the red form of the dye donates a proton to the ionizable

groups on the protein, which disrupts its three dimensional conformation [1]. This unfolding of the

protein exposes the hydrophobic groups within the protein, and the hydrophobic groups interact

non-covalently with the non-polar region of the dye. This also positions the positive amino groups

in the protein interact ionically with the negative portion of the dye, further strengthening their

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Grant Akalonu Bradford & BSA

interaction [1]. The binding of the protein stabilizes the blue form of the Coomassie dye; thus the

amount of the complex present in solution is a measure for the protein concentration, and can be

estimated by use of an absorbance reading. The binding of the protein stabilizes the blue form of

the dye. Therefore, the amount of the complex present in solution can be used to determine protein

concentration, and can be estimated by an absorbance reading [2].

Figure 1: Structure of Coommasie Brilliant Blue G-250

The BCA (bicinchoninic acid assay) can be used to determine the total concentration of

protein in a solution. A color change in the sample occurs from green to purple in proportion to

the total amount of protein present. Therefore, the amount of protein present in solution can be

determined by measuring absorbance, like the Bradford assay. A BCA solution contains the

following reagents at a very high pH: bicinchoninic acid, sodium carbonate, sodium bicarbonate

sodium tartrate, cupric and sulfate pentahydrate [1]. There are two key reactions in the BCA assay.

One is the reduction of Cu2+ to Cu1+in cupric sulfate by the peptide bonds in the protein [3]. This

reaction is important, because the amount of Cu2+ reduced is proportional to the concentration of

protein. The other reaction is a chelation involving two molecules of bicinchoninic acid, and two

Cu+1 ions. This forms a purple colored solution that absorbs light at 562 nm. [3]. The structure of

the BCA reagent and a summary of the two main reactions is shown in Figure 2.

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Grant Akalonu Bradford & BSA

Figure 2: Stucture of BCA reagent, bicinchoninic acid: & summary of the Two Main Reactions in BCA Assay.

Materials & Methods

1) BCA Assay

First, the Bovine Serum Albumin stock solution was prepared by dissolving 8.3mg of BSA in

16mL of distilled water for a concentration of 500g/mL. Then, a series of serial dilutions were

carried out as shown in Table 1.

Tube Water (L) BSA Stock (L) Final BSA

Concentration
(g/mL)
1 800 0 0
2 792 8 5
3 760 40 25
4 720 80 50
5 600 200 125
6 400 400 250
Table 1: Serial Dilutions Prepared in BCA Assay.

BCA working reagent was made by mixing 50mL of reagent A with 1mL of reagent B. Then,

a new set of 6 test tubes were labeled 1 to 6. First, to each test tube, 100L of the corresponding

tube number from Table 1 was added. Next, 2 mL of BCA working reagent solution was added

to each test tube and incubated at 60C for 30 minutes (Figure 3A). When the colors were fully

developed in each tube, the test tube was allowed to cool at room temperature. The absorbance of

all samples was measured using spectrophotometer set at 562nm.

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Grant Akalonu Bradford & BSA

Figure 4. BSA Tubes 1-6 from right to left

2) Bradford Assay

Six tubes consisting of serial dilutions of the BSA stock, water, Bradford reagent, and BSA stock

were prepared. The tubes were prepared as shown in Table 2. After these tubes were prepared, the

absorbance was measured using a spectrophotometer at 595nm.

Tube BSA Stock Water (L) Bradford Total Volume Final BSA
(L) (L) (L) Concentration
(g/mL)
1 0 800 200 1000 0
2 10 790 200 1000 5
3 20 780 200 1000 10
4 30 770 200 1000 15
5 40 760 200 1000 20
6 50 750 200 1000 25
Table 2. Bradford Assay Serial Dilutions.

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Grant Akalonu Bradford & BSA

Figure 5. Bradford Assays Before Analysis on Spectrophotometer.

Results

1) BCA

BCA Sample Final BSA Absorbance

Concentration at 562nm
1
0.9
(g/mL)
y = 0.0038x
0.8 R = 0.9931
1 0 0
0.7 2 5 0.305
Absrobance

0.6 3 25 0.372
0.5 4 50 0.548
0.4
5 125 0.959
0.3
0.2
6 250 1.223
0.1
0
0 50 100 150 200 250 300
Concentration of BSA (mg/mL)

Figure 6: BCA Graph.

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 Grant Akalonu Bradford & BSA

2) Bradford Assay

Bradford Assay
1.4 Sample Final BSA Absorbance
1.2 Concentration at 595nm
y = 0.046x (g/mL)
1 R = 0.96012
Absorbance

1 0 0
0.8
2 5 0.305
0.6 3 25 0.372
0.4 4 50 0.548
5 125 0.959
0.2
6 250 1.223
0
0 5 10 15 20 25 30
Concentration of BSA (g/mL)

Figure 7: Bradford Assay.

Discussion & Conclusion

The Bradford and BCA assays both use the relationship between absorbance and

concentration to determine determine the amount of protein in solution, as demonstrated by Beers

Law. Beers law states that absorbance is proportional the product of the extinction coefficient, the

path length of the cuvette containing the solution, and the concentration of the solution. The slopes

of the graphs in Figures 6 & 7 represent the extinction coefficient of BSA, which is determined to

be 0.0037 mL.g-1.cm-1, and 0.0472 mL.g-1.cm-1 for the BCA and Bradford respectively. The

accuracy with which either test determines the concentration of proteins in solution can be assessed

by looking the value for R2. This value was higher for the BSA, which indicates that the BSA is

more accurate at determining the concentration of protein. This is a sensible result, because the

reactions the color changing agents in BSA is less specific in its method of interaction with

proteins. The Coommasie Brilliant Blue G-250 in the Bradford reagent interacts strongly with

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Grant Akalonu Bradford & BSA

aromatic and basic side chains, indicating that the degree that they bind to proteins depends on the

percentage of basic and aromatic amino acids in the protein. The reagents in the BCA reaction do

not have this restriction, and can bind to a wider variety of proteins in solution.

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Grant Akalonu Bradford & BSA

References

1) http://www.ruf.rice.edu/~bioslabs/methods/protein/protcurve.html
2) https://en.wikipedia.org/wiki/Bicinchoninic_acid_assay
3) https://en.wikipedia.org/wiki/Bradford_protein_assay