Estimation of Protein by Lowry’s Method | Plants

In this article we will discuss about the Lowry’s method for estimation of protein in plants.

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 tartrate are measured in the Lowry’s method.

Materials:

i. 2% Sodium Carbonate in 0. 1N Sodium Hydroxide (Reagent A).

ii. 0.5% Copper Sulphate (CuSO₄. 5H₂O) in 1% potassium sodium tartrate (Reagent B).

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

iv. Folin-Ciocalteau Reagent (Reagent D): Reflux gently for 10 hours a mixture consisting of 100 g sodium tungstate (Na₂WoO₄. 2H₂O), 25 g sodium molybdate (Na₂MoO₄. 2H₂O), 700 ml water, 50 ml of 85% phosphoric acid, and 100 ml of concentrated hydrochloric acid in a 1.5 1 flask. Add 150 g lithium sulfate, 50 ml water and a few drops of bromine water. Boil the mixture for 15 min without condenser to remove excess bromine. Cool, dilute to 1 1 and filter. The reagent should have no greenish tint (Determine the acid concentration of the reagent by titration with IN NaOH to a phenolphthalein end-point.).

v. Protein Solution (Stock Standard): Weigh accurately 50 mg of bovine serum albumin (Frac­tion V) and dissolve in distilled water and make up to 50 ml in a standard flask.

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

Test Procedure:

Extraction of Protein from Sample:

Extraction is usually carried out with buffers used for the enzyme assay. Weigh 500 mg of the sample and grind well with a pestle and mortar in 5-10 ml of the buffer. Centrifuge and use the supernatant for protein estimation.

Estimation of Protein:

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

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

3. The volume to 1 ml in all the test tubes was made. A tube with 1 ml of water serves as the blank.

4. 5 ml of reagent C to each tube including the blank is added and mixed well and allow to stand for 10 min.

5. Then 0.5 ml of reagent D is added and mix well and incubate at room temp, in the dark for 30 min. Blue colour is developed.

6. The readings at 660 nm. is taken.

7. A standard graph is drawn and calculate the amount of protein in the sample.

Calculation:

Express the amount of protein as mg/g or 100 g sample.