Protein Estimation by Lowry's Method PDF

# 3.13 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. Hydrolysing the protein and estimating the amino acids alone will give the exact quantification. The method...

# 3.13 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. Hydrolysing the protein and estimating the amino acids alone will give the exact quantification. The method developed by Lowry *et al*. is sensitive enough to give a moderately constant value and hence largely followed. Protein content of enzyme extracts is usually determined by this method. ## Principle 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. ## Materials - 2% Sodium Carbonate in 0.1N Sodium Hydroxide (Reagent A) - 0.5% Copper Sulphate (CuSO₄.5H₂O) 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 (Na₂WO₄.2H₂O), 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 15 min 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) - Working Standard ## Procedure ### 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 1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1mL of the working standard into a series of test tubes. 2. Pipette out 0.1mL and 0.2mL of the sample extract in two other test tubes. 3. Make up the volume to 1mL in all the test tubes. A tube with 1mL of water serves as the blank. 4. Add 5mL of reagent C to each tube including the blank. Mix well and allow to stand for 10 min. 5. Then add 0.5mL of reagent D, mix well and incubate at room temp in the dark for 30 min. Blue colour is developed. 6. Take the readings at 660nm. 7. Draw a standard graph and calculate the amount of protein in the sample. ## Calculation Express the amount of protein mg/g or 100g sample.