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Biochemistry Laboratory Formal Report

Protein Assay by the Bradford
Method
Arfeille Jan A. Domingo, *Racel Anjelica I. Evangelista,
Roen Saul M. Franco, J-Jen Gagua
Department of Psychology, College of Science

*Corresponding author; e-mail: racelanj@gmail.com
Abstract
The Bradford assay method provides numerous advantages over other
methods as it is based on the protein binding of a dye. It is an analytical
procedure used to measure concentration of a protein in a solution. A
series of test tubes were prepared, one containing different ratios
containing the distilled water and the BSA stock solution and the other
containing 1mL of the unknown protein solution. Both test tubes were
added with 5mL of the Bradford reagent. After the absorbance at 595 nm
of each test tube was determined using the spectrophotometer, the
albumin standard curve was constructed by plotting A
595
versus the
concentration of the BSA and the unknown protein solution was
determined.
Keywords: bradford method, protein, spectrophotometer, BSA standard (bovine serum
albumin)


Introduction
In the determination of an unknown protein solution, there have been several techniques
developed. A time consuming but rather accurate method for the determination of protein
concentration is by acid hydrolyzing a portion of the sample and then it amino acid analysis of
CHEMISTRY
600L
EXPT 3
PAGE 8-9
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the hydrolyzate is carried on. No method in the determination of protein concentration is perfect
because each is dependent on the amino acid content of the protein.
The most commonly used assays are the Biuret and Lowry Assay, BSA Assay,
Spectrophotomeric Assay, and the Bradford Assay. In using the Biuret and Lowry Assay,
substances containing two or more peptide bonds react with the biuret reagent and alkaline
copper sulfate, a purple complex is then formed. The colored product is the result of peptide
nitrogen atoms reacting with Cu
2+
. This method is difficult to do because the standard used, the
purified preparation of the protein to be assayed, is rarely available so the experimenter must
find a relative standard that will yield the same color yield. Measurements of absorbance at
540nm (A
540
) are made against a blank containing biuret reagent or water. The biuret assay has
several advantages, it is quick to do, there are proteins that can replace the standard, and there
are few interfering substances. However, the biuret assay lacks sensitivity.
The Lowry assay is considered to be one of the more sensitive assays because it can
detect protein levels as low as 5. Its procedure is similar to that of the biuret assay except that
a second reagent, Folin-Ciocalteu, is added to increase the amount of color development. Its
greatest advantage is that its sensitivity is a hundred times greater than that of the biuret assay.
There is more time required for the Lowry assay because it should only be used for measuring
changes in protein concentration, not determining the values of protein concentration.
The BCA protein assay is based on the chemical principles similar to what was used in
the Biuret and Lowy assay. The protein to be analyzed is reacted with Cu
2+
and bicinchoninic
adic. The Cu
+
is chelated by BCA, which converts the apple-green color of the free BCA to the
purple color of the copper BCA-complex. This assay has the same sensitivity level as the Lowry
assay and its main advantages are its simplicity and its usefulness in the presence of 1%
detergents.
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In using the Spectrophotomeric assay, the intense ultraviolet light absorption that most
proteins have is centered at 280 nm. This is due to the presence of tyrosine and tryptophan
residues in the protein. A protein solution is transferred to a quartz cuvette and the A
280
is read
against a single reference cuvette containing the protein solvent only. It is particularly suited to
the rapid measurement of protein elution from a chromatography column, where only protein
concentration changes are required. This was employed in the current experiment.
The method of determining the protein concentration of the unknown protein solution
used in this experiment is the Bradford assay. It is based on the protein binding of Coomassie
Brilliant Blue dye that causes a shift in wavelength of maximum absorption of the dye from 465
nm to 595 nm. This method is rapid and has only little interference by nonprotein substances.
Because of its many advantages, biochemical laboratories have adopted the Bradford assay.
In this experiment, its main objective is to determine the concentration of the unknown
protein solution.
Results and Discussion
In this experiment, the Bradford method is commonly used in the determination of the total
protein concentration of an unknown sample. Using the linear regression method, the slope and
y-intercept was determined.
Table 1. The volume (mL) of the BSA stock solution, distilled water (mL), Bradford reagent (mL),
the Concentration, and the Absorbance of S
1
to S
8
.
Solution Blank S
1
S
2
S
3
S
4
S
5
S
6
S
7
S
8
Distilled H
2
O 1.0 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.0
BSA 0.0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 1.0
Concentration 0 40 60 80 100 120 140 160 180
Absorbance 0.001 0.215 0.267 0.293 0.411 0.346 0.509 0.541 0.475

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Table 2. The volume (mL) of the BSA stock solution, distilled water (mL), Bradford reagent (mL),
the Concentration and Absorbance of the unknown protein solution.
Solution Unknown
Distilled H
2
O -
BSA 1.0
Concentration 200
Absorbance 0.433

COMPUTATIONS
A = 0.159 ; B = 1.98 x 10
-3
y = A + Bx
y = 0.159 + 0.00198x
Concentration of Unknown:
Substitute (y = 0.433)
0.433 = 0.159 + 0.00198x
X = 0.433 0.159
0.00198
X = 138.38 g/mL





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Graph 1. Standard curve for BSA, Absorbance (nm) versus Concentration (g/mL)


Experimental Methodology
A set of standards (S
2
- S
8
) were mixed with 5mL Bradford reagent and were prepared by
the groups. The members of this group was only tasked to prepare S
4
. For S
4
, 0.5 mL of the
BSA stock solution was used. Distilled water is then added to bring the volume to 1 mL. For the
unknown protein solution, 1.0 mL of it was mixed with 5 mL of Bradford reagent in a separate
test tube.
Table 3. The volume (mL) of each test tube, BSA stock Solution (mL), distilled water (mL),
Bradford reagent (mL)
Solution S
4
Distilled H
2
O 0.5
BSA 0.5
Concentration 100
Absorbance 0.411

0
0.2
0.4
0.6
0 20 40 60 80 100 120 140 160 180 200
A
b
s
o
r
b
a
n
c
e

Concentration (mg/mL)
Concentration versus Absorbance
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Both test tubes were placed under a vortex mixer for approximately 10-20 seconds
before it was placed under the spectrophotometer. The spectrophotometer was zeroed using S
1

as the reagent blank. After five minutes but before one hour, the absorbance of the standards
and the unknown protein solution at 595 nm (A
595
) were determined. A standard curve was
plotted by using A
595
versus the concentration of the BSA. The concentration of the protein
solution was then calculated in comparison with the standard curve of the BSA.














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