EXPERIMENT: 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 potassium tartarate
4. Folin Ciocaltaeu reagent
5. Bovine serum albumin (BSA) 20 mg/ml
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

1
Experimental Procedures

A. Lowry’s Method
1. Prepare Solution A by adding 0.5% (w/v) of copper sulphate in 1% (w/v)
sodium potassium tartarate.
2. Prepare Solution B by adding 2% (w/v) of sodium carbonate in 0.1 M
sodium hydroxide.
3. Prepare Solution C by adding 50 ml of Solution A to 1.0 ml of Solution B
4. Dilute the BSA solution to 100 g/ml

Set up the following tubes:

Tube
1 2 3 4 5 6
Additions
BSA ml 0 0.2 0.4 0.6 0.8 1
Water ml 2.5 2.3 2.1 1.9 1.7 1.5
Solution C
ml 5 5 5 5 5 5

5. Add 0.5 ml 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 750 nm

B. Bradford Method
1. Prepare the Bradford reagent by dissolving 100 mg of Coomassie Brilliant
Blue G-250 in 50 ml of 95% ethanol. Add 100 ml of 85% (w/v) phosphoric
acid. Dilute the mixture to1000 ml 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
Set up the following tubes

Tube
1 2 3 4 5 6
Additions
BSA (ml) 0 0.2 0.4 0.6 0.8 1
Water (ml) 1 0.8 0.6 0.4 0.2 0
Bradford reagent (ml) 5 5 5 5 5 5

2. Measure the absorbance at 590 nm.

Points to consider:
1. What are the chemical reaction involves in each assay method?
2. Compare the results from the two methods and check its reliability and
accuracy.
3. How can the accuracy of the assays be improved?