Lab Activity 8 Biuret Method IUG, Fall 2012 Dr. Tarek Zaida

Methods Used for Protein Determination Biuret Test Folin-Ciocalteu ( Lowry ) Assay Bicinchoninic Acid ( BCA ) Assay Dye-Binding ( Bradford ) Assay Ultraviolet Absorbance

Biuret Method Peptide Chains Biuret Complexes ( purple color ) Gornall, AG, CS Bardawill, and MM David. J. Biol. Chem. 177: 751. 1949. Layne, E. Spectrophotometric and Turbidimetric Methods for Measuring Proteins. Methods in Enzymology 10: 447-455. 1957. Robinson, HW and CG Hogden. J. Biol. Chem. 135: 707. 1940. Slater, RJ (ed.). Experiments in Molecular Biology. Clifton, New Jersey: Humana Press, 1986. P. 269. Weichselbaum, TE. Am. J. Clin. Pathol. Suppl. 10: 40. 1946.

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

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.

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.