Protein concentration determination using Bradford assay

2A-PH, Group 2, Ballada, T., Banares, S., Belen, R., Bergonia R., Caraig, R.Z., and Cana, N.
Department of Pharmacy, Faculty of Pharmacy, University of Santo Tomas,
Espaa Boulevard, 1015 Manila, Philippines
ABSTRACT
Identification of a substance by its protein concentration can be done
by spectrophotometry and Bradford assay, which includes combining
Coomassie G-250 dye with protein solutions. Six test tubes were prepared
with different volume of standard and
water. After subjecting to a
spectrophotometer, the absorbance of each sample was determined. The
dye used changed to darker colors when in contact with a higher protein
concentration, a higher absorbance meant a higher concentration.
Correlation between absorbance and concentration is determined, R2 is
equal to 0.9721 Also, the line of best fit was calculated, y = 0.0153x +
0.0052.

INTRODUCTION
The Bradford assay is a fast and
fairly accurate method for the estimation of
protein concentration.

The method is based on the

proportional binding of the dye Coomassie
to proteins. The more protein present, the
more Coomassie binds. Furthermore, the
assay is colorimetric, as the protein
concentration increases, the color of the test
sample becomes darker. Coomassie absorbs
at 595 nm. The protein concentration of a
test sample is determined by comparison to
that of a series of protein standards known to
reproducibly exhibit a linear absorbance
profile in this assay. [1]

Figure 1. Structure of Coomassie Brillian

Blue G-250
To calculate absorbances of the
standards and the unknown samples,
spectrophotometry
is
used.
A
spectrophotometer
consists
of
a
spectrometer to produce light of a specific
color and a photometer to measure the
intensity of the light [2]. Therefore, if a
container (or more formally, a cuvette) of
liquid of some opacity is placed between the
spectrometer and the photometer, the
photometer reader would change depending
on the amount of light able to pass through
the cuvette and the absorbance level could
be recorded.

METHODOLOGY
A series of test tube were prepared as
follows:
1
2
3
4
5
6
mL
0
0.2 0.4 0.6 0.8 1.0
standar
d
mL H2O 1.5 1.3 1.1 0.9 0.7 0.5
Table 1. Different volumes of standard and
voumes of H2O in different test tubes
The BSA standard was diluted to 10
mcg/mL. Then, 1.5 mL of Bradford reagent
was added to the individual test tube and
was allowed to stand for 5 minutes. The
samples were transferred into individual
cuvettes and were inserted to the
spectrophotometer. At, 595 nm, the
absorbance was read. The data was gathered.
RESULTS AND DISCUSSIONS
The results in Table 2 shows the data
gathered in Bradford assay after undergoing
the UV-vis spectrophotometer.
mL [BSA]
BSA ug/mL ABS
0.0 0.000 0.000
0.2 1.33 0.032
0.4 2.67 0.052
0.6 4.00 0.061
0.8 5.33 0.080
1.0 6.67 0.112

Table 2. Concentration and Absorbance

using UV-vis Spectrophotometer
The concentration seen in Table 2
was determined using the formula C1V1 =

C2V2 where C is the concentration and V is

the volume.

Concentration vs. Absorbance

0.15
0.1
0.05
0

Figure 3. Graph showing the absorbance and

concentration of the samples
After finding the absorbances of the
standards shown in table 2, we plotted the
concentrations of the standards versus their
absorbances shown in figure 3. The R2 value
of 0.9721 indicated a strong correlation
between

the

concentration

and

the

absorbance. We use this data to calculate the

protein concentrations of the samples. By
using computer software, we were also able
to calculate the equation of the line of best
fit which is y = 0.0153x + 0.0052.
CONCLUSION
The higher the concentration of
BSA, the greater is the absorbance. The
result is more reliable if the correlation
between concentration and absorbance is
greater. Figure 3 shows that the curve line
formed by the results is close in structure to

the drawn straight line which tells us that the

result is close to the expected output.

REFERENCES
Crisostomo, A. & et. al. (2010). Laboratoy
General Biochemistry. Quezon City: C & E
Publishing, Inc.

Ernst, O. (2012). Bradford protein assay.

http://www.ncbi.nlm.nih.gov/pmc/articles/P
MC3164080/
Olson, B. (2013). "Bradford Assay. UNL
classroom server Biochemistry protocols."
http://wwwclass.unl.edu/biochem/protein_as
say/bradford_assay.htm