1. Lab Activity 5 Biuret Method
IUG,
Dr. Tarek Zaida

2. Biuret Method
Peptide Chains
Biuret Complexes ( purple color )

3. Reagents & Instruments
Bovine Serum Albumin (BSA) as a protein standard solution (2mg/ml) in distilled water.
Biuret reagent: dissolve 1.5 g CuSO4.5H2O , 6.0 g of NaKC4H4O6. 4H2O in 500 ml distilled water. Add 300 ml 10% NaOH under continuous swirling. Dilute to 1 L with distilled water and store in a polyethylene bottle.
6 test tubes
Plastic cuvettes
Any protein solution of unknown concentration
Spectrophotometer

4. Spectrophotometer

5. Spectrophotometer

6. Spectrophotometer

7. Cuvettes

8. Procedure
1. Label two sets of tubes with the numbers 1, 2, 3, 4, 5, and 6 for preparation of a standard curve. 2. Label two tubes for the Unknown Protein. 3. Pipette distilled water and Protein Standards solution into the tubes as indicated in table below. 4. Add 1ml Biuret Reagent to each tube and mix thoroughly.

9. 5. Incubate the tubes for at least 20 minutes at room temperature.
6. In the meantime, turn on the spectro-photometer to allow it to warm up. Adjust the wavelength to 540 nm.
7. Add 1 ml distilled water to a cuvette to zero the absorbance of the spectrophotometer.
8. Measure the absorbance of each tube and record the value in the results section.