Lab Manual
CBB20303 BIOCHEMISTRY
EXPERIMENT 2:
Colorimetric Analysis for Protein
Pre-lab exercise:
Discuss the importance of biochemical analysis in bioprocess
technology. Protein is one of the important compounds in bioprocessing and routinely
analysed.
Aim: to learn the methods used in the determination of protein by the colorimetric
analysis. Two methods will be carried out: Lowry’s method and Bradford’s method.
Apparatus and chemicals
1. Copper sulphate
2. Sodium carbonate
3. Sodium tartarate
4. Folin Ciocaltaeu reagent
5. Bovine serum albumin (BSA)
6. Coomassie Brilliant Blue G-250
7. 95% ethanol
8. Phosphoric acid
9. Six 10ml volumetric flasks
10. Four 500-ml beakers
11. 10 test tubes and test-tube rack
12. Spectrophotometer
Experimental Procedures
A. Lowry’s Method
1. Prepare Solution A by adding 0.5% (w/v) of copper sulphate in 1 %( w/v) sodium
tartarate.
2. Prepare Solution B by adding 2% (w/v) of sodium carbonate in 0.1M sodium
hydroxide.
1
Lab Manual
CBB20303 BIOCHEMISTRY
3. Prepare Solution C by adding 50ml of Solution A to 1.0 ml of Solution B
4. Add about 0.2ml of the sample to 2.3 ml of distilled water, followed by 5ml of
Solution C. Allow the mixture to stand at room temperature for 10min.
5. Add 0.5ml of the Folin-Ciocalteau reagent which is prepared by adding 1 part of
the reagent to 2 parts of distilled water.
6. Vortex the mixture and allow to stand at room temperature for 30 mins.
7. Determine the optical density of the reaction mixture at 750nm
8. Prepare the standard curve for protein using BSA of concentration in the range of
0-100mg/L (0, 20, 40, 60, 80 and 100) by repeating step 1-6.
9. Use the standard curve to determine the unknown concentration (i.e. a,b) of the
protein.
B. Bradford Method
1. Prepare the Bradford reagent by dissolving 100mg of Coomassie Brilliant Blue G250 in 50ml of 95% ethanol. Add 100ml of 85% (w/v) phosphoric acid. Dilute the
mixture to1000ml and when the dye completely dissolved, filter through the
Whatman#1 filter paper just before use. (Note: This step has been done for you).
2. Prepare a stock solution bovine serum albumin of 200 μg/ml
3. Prepare a serial dilution on the stock protein solution with distilled water from 20200 μg/ml (0, 10, 30, 50, 70, 100, 120, 150, 180 and 200) of the original stock
concentration. Perform the dilution in duplicate.
4. Add 1.0ml of the diluted solution of the serum albumin in the test tube. Similarly
add the dilution protein sample in the test tube.
5. To all the test tubes, add 5.0ml of Bradford reagent and incubate for 5 mins at
room temperature. Measure the absorbance at 590nm.
6. Plot the absorbance of the standard protein (BSA) against the concentration. Use
the standard curve to determine the unknown concentration of the protein sample.
Report
1. Compare the results from the two methods.
2. Check the reliability and accuracy of the methods
2