Protein estimation by Lowry’s method
Protein can be estimated by different methods as described by Lowry and also by estimating the total nitrogen content. No method is 100% sensitive. Hydrolyzing the protein and estimating the amino acids alone will give the exact quantification. The method developed by Lowry et al1 is sensitive enough to give a moderately constant value and hence largely followed. Protein content of enzyme extracts is usually determined by this method.
The blue color 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 color developed by the biuret reaction of the protein with the alkaline cupric tartrate are measured in the Lowry’s method.
» 2% Sodium Carbonate in 0.1N Sodium Hydroxide (Reagent A)
» 0.5% Copper Sulphate (SuSO4.5H2O) in 1% potassium sodium tartrate (Reagent B)
» Alkaline Copper Solution: Mix 50mL of A and 1mL of B prior to use (Reagent C)
» Folin-Ciocalteau Reagent (reagent D) – Reflux gently for 10 hours a mixture consisting of 100g sodium tungstate (Na2WoO4.2H2O), 25g sodium molybdate (Na2MoO4.2H2O), 700mL water, 50mL of 85% phosphoric acid, and 100mL of concentrated hydrochloric acid in a 1.5L flask. Add 150g lithium sulfate, 50mL water and a few drops of bromine water. Boil the mixture for 15min without condenser to remove excess bromine. Cool, dilute to 1L and filter. The reagent should have no greenish tint. (Determine the acid concentration of the reagent by titration with 1N NaOH to a phenolphthalein end-point).
» Protein Solution (Stock Standard)
Weigh accurately 50mg of bovine serum albumin (Fraction V) and dissolve in distilled 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 200mg protein.
Extraction of Protein from Sample
Extraction is usually carried out with buffers used for the enzyme assay. Weigh 500mg of the sample and grind well with a pestle and mortar in 5-10mL of the buffer. Centrifuge and use the supernatant for protein estimation.
Estimation of Protein
Express the amount of protein mg/g or 100g sample.
1. Lowry, O H, Rosebrough, N J, Farr, A L and Randall, R J (1951) J Biol Chem 193 265.
2. Mattoo, R L (1970) India J Biochem 7 82.
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