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Lab Activity 10

Lab Activity 10. IUG , 2013 Dr. Tarek M.. Zaida. Methods Used for Protein Determination. Biuret Test Folin-Ciocalteu ( Lowry ) Assay Bicinchoninic Acid ( BCA ) Assay Dye-Binding ( Bradford ) Assay Ultraviolet Absorbance. Biuret Test. Peptide Chains.

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Lab Activity 10

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  1. Lab Activity 10 IUG, 2013 Dr. Tarek M.. Zaida

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

  3. Biuret Test 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.

  4. Biuret Test • Advantages • Reproducible • Very few interfering agents • Fewer deviations than with the Lowry or ultraviolet absorption methods Disadvantages • Requires large amounts protein (1-20mg) • Low sensitivity

  5. 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. • 12 test tubes • Plastic cuvettes • Any protein solution of unknown concentration • Spectrophotometer

  6. Procedure • Label two sets of tubes with the numbers 1, 2, 3, 4, 5, and 6 for preparation of a standard curve. • Label two tubes for the Unknown Protein. • Pipette distilled water and Protein Standards solution into the tubes as indicated in Table below. • Add 1ml Biuret Reagent to each tube and mix thoroughly. • Incubate the tubes for at least 20 minutes at room temperature. • In the meantime, turn on the spectrophotometer to allow it to warm up. Adjust the wavelength to 540 nm. • Add 1 ml distilled water to a cuvette to zero the absorbance of the spectrophotometer. • Measure the absorbance of each tube and record the value in the results section.

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