Estimation of Protein by colorimetric method.

 To estimate the concentration of protein in a given sample by the Bradford method.

 Requirements

Chemicals

BSA, distilled water, Coomassie brilliant blue, ethanol, orthophosphoric acid, monobasic sodium phosphate, dibasic sodium phosphate

Equipment

·        Weighing balance,

·        pH meter,

·        spectrophotometer

·        Glassware

·        Test tubes,

·        conical flasks

·        glass pipette

·        beaker

Solutions

Bradford Reagent

1. Take 100 mg Coomassie brilliant blue in an airtight bottle and dissolve in 50 ml ethanol  (95 %). Incubate for an hour at room temperature.

2. After incubation, slowly add 100 ml of orthophosphoric acid (85%) and adjust the final volume to 1000 ml using DW.

3. Filter the reagent using Whatman filter paper no. 1 and store it in an amber coloured bottle, which is stable for maximum of 3 weeks.

0.2 M Phosphate buffer (pH 7.5)

Standard Protein: 0.02 mg/ml solution of BSA

Procedure

1. Warm-up spectrophotometer 15 min before use.

2. Take 9 clean glass test tubes. Into each test tube, carefully pipette one of the following  volumes of a 20 mg/ml solution of BSA: 0.0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 ml. In the last two test tubes put 1 ml of test samples each as duplicates.

3. Bring the total volume of liquid in all tubes to 2.0 ml by adding an appropriate amount of phosphate buffer.

4. Add 2 ml of Bradford reagent in all tubes, vortex thoroughly and incubate at room temperature for 15 min at room temp.

5. Determine the OD of each sample at 595 nm. Plot the standard curve using the concentration of standard tubes of BSA (mg/ml) against the absorbance.

6. Calculate the mean of absorbance of the duplicate sample and find out the protein concentration present in the given sample from standard graph and formula (given below) and express in mg/ml.

 

Sr. No.	Std. BSA (ml)	Sample (ml)	Phosphate Buffer (ml)	Bradford’s Reagent (ml)	OD at660 nm
1	--	--	2.0
2	0.1	--	1.9
3	0.2	--	1.8
4	0.4	--	1.6
5	0.6	--	1.4
6	0.8	--	1.2
7	1.0	--	1.0
8	--	1	1.0
9	--	1	1.0

Calculations The concentration of protein (% mg) in the given sample is:

 

The concentration of protein in the given sample estimated with Bradford’s method is found to be _______ mg/ml.