低活力蛋白酶活力测定方法.doc

Universal Protease Activity Assay: Casein as a Substrate Written Protocol: A. Abstract Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases, which is what we do during our quality control procedures. In this assay, casein acts as a substrate. When the protease we are testing digests casein, the amino acid tyrosine is liberated along with other amino acids and peptide fragments. Folin & Ciocalteus Phenol, or Folin’s reagent primarily reacts with free tyrosine to produce a blue colored chromophore, which is quantifiable and measured as an absorbance value on the spectrophotometer. The more tyrosine that is released from casein, the more the chromophores are generated and the stronger the activity of the protease. Absorbance values generated by the activity of the protease are compared to a standard curve, which is generated by reacting known quantities of tyrosine with the F-C reagent to correlate changes in absorbance with the amount of tyrosine in micromoles. From the standard curve the activity of protease samples can be determined in terms of Units, which is the amount in micromoles of tyrosine equivalents released from casein per minute. B. Materials Reagents:Protease ( P4630) Potassium Phosphate, Dibasic, Trihydrate ( P5504) Casein ( C7078) Trichloroacetic Acid ( T0699) Folin & Ciocalteu’s Phenol Reagent ( F9252) Sodium Carbonate, Anhydrous ( S2127) Sodium Acetate, Trihydrate ( S8625) Calcium Acetate ( C1000) L-Tyrosine, Free Base ( T3754) Equipment:0.45 um polyethersulfone syringe filter and syringe 0.45μm Dram vials or polypropylene tubes capable of holding 15 mls of solution Spectrophotometer Cuvettes Pipettes Stir/Hot plate Stir bar ScalepH Meter Graphing Program C. Preparation of Reagents Before beginning the assay, we need to make sure that t