Practical Biochemistry

Lab Report
BRADFORD PROTEIN ASSAY

GROUP. NUMBER CLASS

04 L1.4.2

STUDENT NAME

BI12-247: Lê Diệu Linh

BI12-313: Hoàng Hoàng Ngân

BI12-260: Nguyễn Thảo Ly

BI12-219: Bạch Vũ Hồng Khôi

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I. Objective:
- Understanding the principle, mechanism and protocol of Brandford methods
- Practicing dilution technique, using spectrophotometer
- Applying the Brandford in determining the concentration of a protein solution

II. Principle:
- The dye Coomassie brilliant blue G-250 absorbing the light maximum at wavelength
465nm
- When mixing this reagent with protein, it binds to the base side chain amino acid
(Histidine, Arginine, Lysine) or aromatic side chain amino acid (Phenylalanine, Tyrosine,
Tryptophan).
- This leads to the change of color into blue which intensity is depended on the
concentration of the protein. And the maximum wavelength to be absorbed change into
595nm.
- Using the spectrophotometer to detect the OD at 595nm of the mixture of a standard
protein with known concentration (here we use BSA) to sketch the graph of concentration
with respect to the OD.
- Using the graph as the reference to determine the concentration of other protein solution.

III. Protocol:
1. Prepare the test tube standard:
- Prepare solution of BSA with the concentration: 1.5 mg/ml, 1 mg/ml, 0.8 mg/ml, 0.6
mg/ml, 0.4 mg/ml, 0.2 mg/ml, 0.1 mg/ml

Volume of
Final
BSA solution Volume Real total
No. of Conc.
of previous of added volume after
tube (mg/ml
higher conc. water(µl) diluted (µl)
)
(µl)
2 390
1 1.5 390 130 520
2 1 360 180 540
3 0.8 360 90 450
4 0.6 270 90 360
5 0.4 200 100 300
6 0.2 120 120 240
7 0.1 75 75 150
8 0 0 150 150

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 (1) Add water with the volume in column 3 respectively in 8 tubes
(2) Take 390 µl of 2mg/ml BSA solution into the tube 1, pipetting to mix up the
mixture
(3) Repeat the procedure until the tube 7
(4) Take 67 µl of each tube twice and put into 2 tubes
(5) Add 2ml of Coomassie dye, mix up and pour into the cuvette
(6) Using spectrophotometer to measure the OD of each tube (measure tube 8 (of
pure water+ Coomassie dye ) to set blank )
(7) Using the results to sketch the graph of concentration with respect to the OD and
find the function

2. Prepare the unknown sample:

- Dilute the sample 8 times and 10 times:
(1) Take 20 µl of sample to dilute with 180 µl to obtain the solution with 10 times
dilution
(2) Take 20 µl of sample to dilute with 140 µl to obtain the solution with 8 times dilution
(3) Then take the 67 µl of each tube twice and put into 2 tubes and add 2ml Coomassie
dye in each tube and measure the OD
- Use the value to find the concentration by the function above

IV. Result:
(1) The Standard test tube:

Measure OD (abs)
Conc. Trial 1 Trial 2 Average OD (abs)
0.1 0.186 0.203 0.1945
0.2 0.405 0.506 0.4555
0.4 0.727 0.628 0.6775
0.6 0.915 1.08 0.9975
0.8 1.524 1.558 1.541
1 1.789 2.054 1.9215

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 Standard Calibration Curve
2.5

2
f(x) = 1.90141666666667 x + 0.00729166666666681
OD (595 nm)

1.5 R² = 0.993993238526841
Series2
1 Linear (Series2)

0.5

0
0 0.2 0.4 0.6 0.8 1 1.2
Concentration (mg/ml)

(2) The unknown sample (A)

- Using equation: y=1.9014x+0.0073

Measure (abs) Averag Concentration

Trial 1 Trial 2 e of sample
10 times 1.047 1.098 1.0725 5.636
8 times 1.345 1.309 1.327 5.579
- The concentration of sample is 5.6 mg/ml

(3) Troubleshooting:
- We can’t measure the OD of 1.5mg/ml BSA solution because the volume retained after
taking 360 µl (to make 1 mg/ml solution) is too small
 We may put less water than required when diluting to create 1.5mg/ml solution
 The concentration of all tube in the serial dilution process might be higher than theory
 This increases the value of OD with respect to the concentration in the reference
 The calculated concentration of unknown sample might lower than reality
- The cuvette is dirty might also increase the value of OD with respect to the concentration
in the reference

V. Alternative methods:
(1) BCA Protein Determination:
Principle:
- First, the peptide bonds in protein reduce Cu2+ ions from the copper(II) sulfate to Cu1+. 
- The amount of Cu2+ reduced is proportional to the amount of protein present in the
solution.

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- Next, two molecules of bicinchoninic acid chelate with each Cu1+ ion, forming a purple-
colored complex that strongly absorbs light at a wavelength of 562 nm.
- Then using spectrometer to determine the concentration of protein similarly as the
Bradford method

(2) Lowry Protein Assay:

Principle:
Similar as BCA Protein Determination ( different in reagents of reactions creating
complex with proteins)