Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes.
A simple inexpensive sensitive protease assay was developed using soluble fluorescein isothiocyanate (FITC)-labeled casein. Casein was reacted with FITC to form the fluorescein thiocarbamoyl derivative. This substrate is cleaved by trypsin, chymotrypsin, elastase, subtilisin, and thermolysin in a linear time-dependent manner. These enzymes can be measured in the nanogram and subnanogram range using this assay. The assay is reproducible, has a low blank, and uses casein, which resembles natural substrates of most proteases.
D000818:Animals; D002364:Caseins; D055598:Chemical Phenomena; D002621:Chemistry; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008264:Macrophages / Q000201:enzymology; D010447:Peptide Hydrolases / Q000032:analysis*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates
D002364:Caseins; D005452:Fluoresceins; D013861:Thiocyanates; C044337:fluorescein thiocarbamoyl casein; D010447:Peptide Hydrolases; D016650:Fluorescein-5-isothiocyanate