EXPERIMENT NO.

09
Aim: To estimate protein content in unknown sample using Bradford’s assay.
Materials Required:
Spectrophotometer, Bovine Serum Albumin (BSA), Cuvette, Pipettes, Pipette tips, Micropipette,
1.5 mL Micro Centrifuge Tubes, Methanol, Milli-Q Water, Bradford’s Reagent, Phosphate buffered
saline with Tween-20 (PBST).
Principle:
The Bradford protein assay is one of several simple methods commonly used to determine the
total protein concentration of a sample. The principle of this assay is that the binding of protein
molecules to Coomassie Blue G250 dye under acidic conditions results in a colour change from
brown to blue. This method actually measures the presence of the basic amino acid residues,
arginine, lysine and histidine, which contributes to formation of the protein-dye complex. Within
the linear range of the assay (~5-25 µg/mL), the more protein present, the more Coomassie Blue
binds. Furthermore, the assay is colorimetric; as the protein concentration increases, the colour of
the test sample becomes darker. Coomassie Blue 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. Although different protein standards
can be used, we have chosen the most widely used protein as our standard - Bovine Serum
Albumin (BSA).
Procedures:
1. Prepare standard protein stock solution of Bovine Serum Albumin (BSA) in Phosphate
buffered saline with Tween-20 (PBST) of concentration 100 mg/mL and store at 4°C.
2. Take 3 micro centrifuge tubes and mark them as 2, 4, and 6 for standard working solutions
of concentration 2 µg/µL, 4 µg/µL and 6 µg/µL respectively.
3. Preparation of standard working solutions:
a) For 2 µg/µL, take MC tube 1 and pipette out 980 µL of PBST in it and then 20 µL of 10%
BSA solution. Hint: use the relation S1V1 = S2V2 where S1 is concentration of BSA solution
(i.e.,100 µg/µL), V1= required volume of 10% BSA solution, S2=concentration of standard
working solution (i.e., 2 µg/µL in this case) and V2=final volume of solution (i.e., 1000 µL)
b) For 4 µg/µL, take MC tube 2 and pipette out 960 µL of PBST in it and then 40 µL of 10%
BSA solution.
c) For 6 µg/µL, take MC tube 3 and pipette out 940 µL of PBST in it and then 60 µL of 10%
BSA solution.
4. Now, take another 5 micro centrifuge tubes and mark them as X for Blank, U for unknown
sample and 2’, 4’, and 6’ for preparation of sample solution for 2 µg/µL, 4 µg/µL and 6 µg/µL
working standard solution respectively.
5. Preparation of sample solutions:
a) In tube ‘X’, pipette out 500 µL of Milli-Q water.
b) In tube ‘2’’, pipette out 498 µL of Milli-Q water and then 2 µL of solution from tube 2.
c) In tube ‘4’’, pipette out 498 µL of Milli-Q water and then 2 µL of solution from tube 4.
d) In tube ‘6’’, pipette out 498 µL of Milli-Q water and then 2 µL of solution from tube 6.
e) In tube ‘U’, pipette out 498 µL of Milli-Q water and then 2 µL of unknown sample.
Now, add 500 µL of Bradford’s Reagent to each tubes (X, 2’, 4’, and 6’) and mix well.
 6. Incubate for at least 5 min.
7. Measure absorbance (OD) at 595 nm.
8. Plot absorbance at 595 nm against the concentration of protein.
9. Find the amount of protein in unknown sample using above plotted standard curve.
Observation Table:
Sl. No. Test Sample Sample Vol. of Milli- Vol. OD at 595 nm
Volume, Q Water, Bradford
µL µL Reagent,
µL
1 Blank -- 500 500 0
2 BSA 2 498 500 (0.303+0.301+0.302)/3
Standard - 2 =0.302
µg/µL
3 BSA 2 498 500 (0.495+0.487+0.490)/3
Standard - 4 =0.4906
µg/µL
4 BSA 2 498 500 (0.702+0.702+0.697)/3
Standard - 6 =0.7003
µg/µL
5 Unknown A 2 498 500 0.183
6 Unknown C 2 498 500 0.600

BSA Standard Curve

0.8

0.7
y = 0.0572x + 0.0298
R² = 0.9886

0.6
OD at 595 nm

0.5

0.4

0.3

0.2

0.1

0
0 2 4 6 8 10 12 14

Concentration of Protein (µg/µL)

Calculations:
From the standard curve, we have
y = 0.0572x + 0.0298
or, OD = 0.0572*(Conc. of Protein) + 0.0298
or, Conc. of Protein = (OD – 0.0298)/0.0572
Hence, conc. of protein in sample A = (OD – 0.0298) / (0.0572 x Vol. of diluted protein used for assay)
= (0.183 – 0.0298) / (0.0572 x 2)

= 1.339 µg/µL
And, conc. of protein in sample C = (OD – 0.0298) / (0.0572 x Vol. of diluted protein used for assay)
= (0.6 – 0.0298) / (0.0572 x 2)

= 4.984 µg/µL
Results:
The concentration of protein in unknown sample A is 1.339 µg/µL and in unknown sample C is
4.984 µg/µL.
Precautions:
1. The Coomassie Blue G250 precipitates out of the solution with time. Bradford reagent
should therefore be filtered through Whatman no. 1 filter paper before use.
2. The dye has preferential binding to lysines and arginines in the proteins but does not bind
to free lysine and arginine.
3. The dye does not bind to the peptides smaller than about 3 kDa; Bradford assay, therefore,
is not suitable for small peptides.
4. UV/Visible spectrophotometer should be switched on at least 15 min before use.
5. Plastic and glassware used in the assay should be absolutely clean and detergent free.
6. Quartz (silica) spectrophotometer cuvettes should not be used, as the dye binds to this
material. Traces of dye bound to glassware or plastic can be removed by rinsing with
methanol or detergent solution.
7. Sensitivity of this assay is high, so use lesser protein for standard and unknown.
8. It is advisable to wash the cuvette when you change the dilution.
Discussion:
Through the experiment, the group was able to solve for the concentration of the unknown
protein solution by using the linear regression method and by plotting the standard curve by
absorbance versus concentration. Using the standard curve, the unknown protein solution had a
concentration of 1.339 µg/µL (A) and 4.984 µg/µL (C). The absorbance obtained for the unknown A
and B are 0.183 nm and 0.600 nm respectively.
The Bradford method shows that the absorbance has a direct relationship with the concentration
of the protein sample, meaning if the absorbance is high, the concentration of the sample is also
high.