Cupp-Enyard Carrie
Sigma Aldrich.
J Vis Exp. 2009 Aug 4(30):1514. doi: 10.3791/1514.
The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate. Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured. The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 microg/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.
蛋白酶荧光检测试剂盒提供即用型试剂，用于检测蛋白酶活性的存在。这种检测蛋白酶活性的简单方法使用异硫氰酸荧光素（FITC）标记的酪蛋白作为底物。蛋白酶活性导致FITC标记的酪蛋白底物裂解成较小的片段，这些片段在酸性条件下不会沉淀。在将蛋白酶样品与底物孵育后，通过加入三氯乙酸（TCA）使反应酸化。然后将混合物离心，未消化的底物形成沉淀，较小的、酸溶性片段留在溶液中。将上清液中和并测量FITC标记片段的荧光。所述试剂盒方法可检测浓度约为0.5微克/毫升（向测定中加入5纳克胰蛋白酶）的胰蛋白酶蛋白酶对照。通过延长孵育时间（最长24小时）可提高这种灵敏度。该测定在微量离心管中进行，并提供了使用比色皿或多孔板进行荧光检测的程序。
