BIURET PROTEIN ASSAY

BIURET PROTEIN ASSAY

MATERIALS

• Biuret Reagent

• Bovine serum albumin (BSA)

• Spectrophotometer and tubes

PROCEDURE

1. Prepare standard dilutions of BSA containing 1, 2.5, 5.0, 7.5 and 10 mg/ml protein. Prepare serial dilutions of the unknown samples.

   
   

2. Add 1.0 ml of each of the standards, each sample, and 1.0 ml of distilled water to separate tubes. Add 4.0 ml of Biuret reagent to each tube. Mix by vortex.

   
   

3. Incubate all of the tubes at 37 ° C for 20 minutes.

   
   

4. Turn on and adjust a spectrophotometer to read at a wavelength of 540 nm.

   
   

5. Cool the tubes from Step 3, blank the spectrophotometer and read all of the standards and samples at 540 nm.

   
   

6. Plot the absorbance of the standards vs their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.

NOTES

The Biuret reaction was one of the first for the determination of protein concentration. It remains as a rapid determination, but is not very accurate. It is useful during protein separation procedures since there are fewer salt interference reactions than with the Bradford or Lowry techniques. The color formed is stable for about 1-2 hours and consequently all spectrophotometer readings must be made as soon as possible after the incubation step.