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Proteases are naturally present in all organisms. These enzymes are involved in a multitude of physiological reactions from simple digestion of food proteins to highly regulated cascades. Proteases can either break specific peptide bonds (limited proteolysis), depending on the amino acid sequence of a protein, or break down a complete peptide to amino acids (unlimited proteolysis). The activity can be a destructive change (abolishing a protein's function); an activation of a function (pre-form to mature form) or it can be a signal in a signaling pathway.

Protease Activity Assay Kit provides a simple, reproducible, and sensitive tool of measuring the activity of a broad spectrum of proteases (e.g. trypsin, chymotrypsin, thermolysin, proteinase K, protease XIV, and elastase) in cell lysates, plasma, serum, and other biological liquid samples. In this assay, fluorescein isothiocyanate (FITC)-labeled casein is used as a general protease substrate. The fluorescein label on the FITC-Casein is highly quenched. Upon digestion by proteases present in the sample the FITC-Casein substrate is cleaved into smaller peptides which abolish the quenching of the fluorescence label. The fluorescence of the FITC-labeled peptide fragments is measured at Ex/Em = 490/525 nm. The increase in fluorescence intensity is directly proportional to protease activity.

• Microplate reader capable of measuring fluorescence intensity at Ex/Em = 490/525 nm
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Tubes to prepare sample dilutions
• 15 mL conical tubes

1. Add 50 µL of samples, positive control, substrate background (just by adding assay buffer) to the 96-well microtiter plate (black bottom).
2. Add 100 µL of standard to the 96-well microtiter plate.
3. Initiate the reaction by adding 50 µL diluted protease substrate solution to each well. Carefully shake the plate for a few seconds to mix.
4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 490/525 nm by kinetic reading at room temperature.