Protease Assay Kit

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  • Published date: July 22, 2021
    • United States

Cat.No. :Kit-2416
Product Overview :The Protease Assay Kit enables easy, fast and reproducible quantitation of protease activity. The homogeneous assay is performed in a microplate and requires no strong acids or separation steps. TPCK trypsin is provided as a general protease standard and may be used for secondary calibration of other specific protease standards. The assay method uses succinylated casein and trinitrobenzenesulfonic acid (TNBSA).1-3 Succinylated casein is native casein that has been treated with succinic anhydride to block primary amines on the surface of the protein. In the presence of protease, the succinylated casein is cleaved at peptide bonds, thereby exposing primary amines (predominantly α-amines). TNBSA reacts with these exposed primary amines to produce an orange-yellow product whose intensity may be measured at 450nm. The increase in color relative to sample without succinylated casein is a measure of protease activity in the sample.1 Because TNBSA produces color when it reacts with any primary amine, it is critical that blanks for each sample be included in the assay to correct for the effect of amines (general protein content) in the protease sample. Amine-containing buffers such as Tris will cause high background, although correction for small amounts of Tris (< 50mM) in the sample can be made using the blank. In practice, when the concentration of amines in the buffer or sample is high, the sample must be diluted so that its blank (no succinylated casein) absorbance at 450nm in the assay is less than 0.500. The assay is compatible with up to 1mM DTT in the protease sample; however, the background (blank) absorbance reading will be increased.
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