To perform the estimation of protein by Biuret method

AIM: To perform the estimation of protein by Biuret method.

PRINCIPLE:
Biuret method is the simplest method for protein estimation. This method is sensitive to the amino acid composition of the protein. Its sensitivity is moderately constant from protein to protein and because of its simple procedure and quick result, it is used to estimate protein in crude extract over a large range of concentration. This method can also be used to monitor the concentration of protein during purification.
This assay is based on copper ions binding to peptide bonds of protein under alkaline conditions to give a violet or purple color. The intensity of the charge transfer absorption bond resulting from the Cu-protein complex is linearly proportional to the mass of protein present in the solution. The chromophore or light-absorbing center seems to be a complex between the peptide backbone and cupric ions.

PROCEDURE:
1) Pipette standard BSA (50 mg/ml) and test sample as indicated in table in duplicates.
2) Adjust the volume to 0.2 mi with distilled water.
3) Add 3 ml of Biuret reagent. Mix and incubate at Room Temperature for 10 minutes.Read the optical density using spectrophotometer at 600 nm or colorimeter using suitable filter and record the readings.

1) Construct a calibration curve by plotting average optical density reading on ‘Y’ axis against std. Protein concentration (in mg) on ‘X’ axis.
2) Record the value ‘X’ from the graph corresponding to the optical density reading for the test sample.
3) Calculate the sample concentration using the following formula.
Protein concentration in test Sample=(X/V)x1000 mg/ml

X= Value from graph in mg
V= Volume of Sample in ml
RESULT: