9 Estimation of protein
 

 

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Protein can be estimated by different methods. No method is 100% accurate. Hydrolysing the protein and estimating the amino acids alone will give the exact quantification. The method developed by Lowry et al (1951) is sensitive enough to give a moderately constant value and hence largely followed.

Principle: The blue colour developed by the reduction of the phosphomolybdic-phosphotungstic components in the Folin-Ciocalteau reagent by the amino acids tyrosine and tryptophan present in the protein plus the colour developed by the biuret reaction of the protein with the alkaline cupric tartarate are measured in the Lowry's method.

 

Materials and Reagents

 

2% Sodium carbonate in 0.1 N Sodium Hydroxide (Reagent A)

0.5% Copper sulphate in 1 % potassium sodium tartarate (Reagent B)

Alkaline copper solution: Mix 50ml of A and 1 ml of B prior to use (Reagent C)

Folin-Ciocalteau reagent

Protein solution (Stock standard)

 

 

Composition of reaction mixtures
 

A. Protein standard stock

Weigh accurately 50mg of bovine serum albumin and dissolve in distiiled water and make up to 50ml in a standard flask.

Working standard: Dilute 10ml of the stock solution to 50ml with distilled water in a standard flask. One ml of this solution contains 200μg protein.

 

Procedure

 

1.   Weigh 500mg of the sample and grind well with a pestle and mortar in 5-10 ml of the buffer.

2.   Centrifuge and use the supernatant for protein estimation.

3.   Pipette out 0.2,0.4,0.6, 0.8 and 1 ml of the working standard into a series of test tubes.

4.   Pipette out 0.1 ml and 0.2ml of the sample extract in two other test tubes.

5.   Make up the volume to 1ml in all the test tubes. A tube with 1ml of water serves as the blank.

6.   Add 5ml of reagent C to each tube including the blank. Mix well and allow to stand for 10min.

7.   Then add 0.5ml of Folin-Ciocalteau reagent, mix well and incubate at room temp in the dark for 30 min. Blue colour is developed.

8.   Take the readings at 660nm.

9.   Draw a standard graph and calculate the amount of protein in the sample. Express the amount of protein in mg/g or 100g sample.

 



LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem. 193(1):265-75 (Click here to download).