title: Use of the protease fluorescent detection kit to determine protease activity.
abstract: 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.
authors.affiliation: Sigma Aldrich.
authors.forename: Carrie
authors.initials: C
authors.lastname: Cupp-Enyard
mesh_terms: D002364:Caseins / Q000737:chemistry*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000032:analysis* / Q000378:metabolism; D011933:Reagent Kits, Diagnostic; D014357:Trypsin / Q000737:chemistry
chemical_list: D002364:Caseins; D005456:Fluorescent Dyes; D011933:Reagent Kits, Diagnostic; D010447:Peptide Hydrolases; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate
references.citation: Anal Biochem. 1984 Nov 15;143(1):30-4
references.pmid: 6442109