QUANTITATIVE PROTEIN ANALYSIS: BRADFORD ASSAY

Hiroaki T.A. Gaviola, Jan D. Gayamo, Claris G. Guiyab,

Sean K. Herman, and Edward O. Jaquiaca
Group 4 2D Medical Technology General Biochemistry Laboratory

ABSTRACT
The Bradford protein assay is a method used to determine the protein concentration of a solution through
spectrophotometric analysis. The experiment aims to accurately solve for the protein concentration of an unknown
sample by plotting a standard curve on the relationship between the standards’ absorbance at 595 nm to the protein
concentration as expressed in the Beer-Lambert Law. 5 standards of different concentrations were prepared by adding
0.1, 0.5, 1.0, 1.5, and 2.5mL of BSA stock solution respectively. Distilled water was added to each standard to raise
their volumes to 2.5mL, and then 2.5mL of Bradford reagent was added to each test tube afterwards. The absorbances
of the samples were determined using a spectrophotometer at 595 nm. A standard curve was then plotted showing the
relationship between the concentration and absorbance of the samples. Using the linear equation derived from the
standard curve, the protein concentration of the unknown sample was determined. The computed protein concentration
of the unknown is 0.028 mg/mL. The absorbance reading of the unknown solution at 595 nm is 0.538.

INTRODUCTION Lambert law, and determine the protein

concentration of the unknown sample using linear
Proteins are one of the four macromolecule regression analysis.
building blocks of life that are composed of one or
more chains of amino acids in a specific order. EXPERIMENTAL
They are needed by the body to function as it A. Samples used
regulates, help function, and facilitate the
structure of the body’s cells, tissues, and even BSA Standard Stock Solution, Distilled Water,
organs. This makes the quantification of a Bradford Reagent, Unknown Sample
protein’s concentration vital in the field of
B. Procedure
biochemistry as even small changes in the
7 test tubes were utilized by the group. The
quantity can noticeably alter the results of
first test tube was a 2.5 mL blank containing only
applications like developing drugs, analyzing
distilled water while the last contained the
potency, etc.
unknown sample. Test tubes 2-6 served as the
The method used to quantify or determine the standards with known protein concentrations and
protein concentration of a sample is called a were prepared by diluting the BSA standard stock
protein assay. There are different ways of finding solution with water. Each standard test tubes
the protein concentration of a sample, but there contained the following:
are three significant methods namely: Biuret total
Table 1. Corresponding Measurements of BSA
protein assay, MicroBradford total protein assay,
and Distilled Water in Each Standard
and Bradford total protein assay. The method that
the group did, and will look in detail to, is the BSA Distilled
Bradford total protein assay. Test Tube Standard Water (mL)
Stock
The Bradford total protein assay method is a Solution
simple procedure of adding a reagent containing (mL)
Coomassie Brilliant Blue G-250 which is a dye that Standard 1 0.10 2.40
reacts uniquely when encountering proteins. This Standard 2 0.50 2.00
method accurately determines the concentration Standard 3 1.00 1.50
of a protein in a solution using spectrophotometric Standard 4 1.50 1.0
analysis. The standards that are to be used will be Standard 5 2.50 0
subjected to a spectrophotometer at 595 nm to
determine their absorbances. A Subsequently, 2.5 mL Bradford reagent was added
spectrophotometer basically measures the amount to each test tube and were left to stand for five
of light that a solution absorbs. minutes.

By the end of the experiment, the students are

expected to understand the Bradford total protein
assay method, draw a standard curve and show
the relation of the samples’ absorbance and
protein concentration in expression of the Beer-
 Table 2. Concentration, absorbances, and
corrected absorbances of the samples
Blan S1 S2 S3 S4 S5 Unkno
k wn
Con 0.00 0.00 0.01 0.02 0.03 0.05 x
c. 2
Abs 0.23 0.39 0.55 0.67 0.81 1.05 0.774
6 7 9 2 1 9
C. 0.00 0.16 0.32 0.43 0.57 0.82 0.538
Abs 1 3 6 5 3

The concentrations (Conc.), absorbances (Abs),

and corrected absorbances (C. Abs) of the
Figure 1. Standards and blank after addition of solutions can be found in table 2. The absorbances
Bradford reagent were corrected using a blank by subtracting the
blank’s absorbance from the standards and the
A microwell plate was then prepared and its unknown. This is done to get the absorbance of
wells labeled according to their corresponding only the proteins in the samples.
content. Each of the 7 solutions was dropped 3
times into their corresponding well. Afterwards,
the microwell plate was subjected to
spectrophotometry. The absorbances of the
solutions were read at 595 nm.

A standard curve was then drawn by plotting the

concentrations of the solutions on the X axis, and
the corrected absorbances of the solutions on the
Y axis, in Microsoft Excel. The protein
concentration was then determined using the
linear equation of the standard curve.

Computation for conc. of unknown sample

𝑦 = 𝑚𝑥 + 𝑏
𝑦 = 13.438𝑥 + 0.1626
0.538 = 13.438𝑥 + 0.1626
0.538 − 0.1626
𝑥=
13.438
RESULTS AND DISCUSSION
The Bradford protein assay is a method used to
determine the protein concentration of a solution. It
is based on the reaction of the acidic solution of
Coomassie Brilliant Blue G-250 when it encounters
proteins. Its maximum absorbance shifts from 465
nm to 595 nm, causing a visible color change from
red to blue, when it binds with proteins. The samples
were read at 595 nm because it is the maximum
absorbance of the Bradford reagent.
Figure 1. Standard curve (BSA conc. vs
corrected absorbance)
The standard curve was determined by inputting
the data of the samples in Microsoft Excel where
the BSA concentration (mg/mL) is plotted on the
X axis, while the corrected absorbances of the
samples are plotted on the Y axis. The
concentration and absorbance of the group’s
unknown can be seen in red in the graph.
The corrected absorbance reading of the
unknown solution at 595 nm is 0.538 while the
computed protein concentration of the unknown
solution is 0.028 mg/mL. Although the
computation is correct, the measured
concentration is not even remotely close to the
true concentration of the sample. This may be
caused by multiple reasons including the addition
of incorrect amounts of the reagent, BSA solution,
and distilled water that may in turn be caused by
the manual pipetting of the mentioned samples.
This will make the absorbance readings
inaccurate. Contamination of the samples may
also have caused the wrong concentration
measured.

The Bradford protein assay method is a fast and

easy method of determining the concentration of
an unknown protein sample. It requires only a
small amount of time to accomplish when a
micropipette is used. This method is also relatively
accessible as it requires a spectrophotometer that
can measure a high wavelength, 595 nm, which
most spectrophotometers can. It is also a sensitive
method as it can measure protein samples as
small as 1-20 μg. However, an inaccurate
measurement of a single sample can lead to an
inaccurate linear equation and absorbance reading
which will in turn cause an inaccurate
measurement of the unknown protein
concentration.

REFERENCES
From the internet (on-line)
[1] He, F. Bradford Protein Assay. Retrieved
from https://bio-protocol.org/bio101/e45 3/20/11
[2]