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Product Detail

Detect changes in intracellular chymotrypsin-like serine protease enzyme activity in whole living cells with the FLISP, Fluorescent-Labeled Inhibitors of Serine Proteases, product line of assay kits. This assay''s FLISP reagent, FLCK, utilizes a leucine (L) chymotrypsin-targeting amino acid residue linked on the amino terminus with a carboxyfluorescein (FAM) fluorescent reporter tag. The carboxyl end of the L amino acid residue contains a chloromethyl ketone (CMK) reactive group that targets the catalytic site of serine proteases.

After quickly penetrating the lipid bilayer membranes of the target cell population, the chymotrypsin-targeting FLISP probe FLCK will interact with the active catalytic sites of chymotrypsin-like proteases, quickly forming covalent bonds with either the reactive site histidine (N-H). Unbound FLISP reagent is easily removed during the wash step, leaving cells with greater quantities of active chymotrypsin-like enzyme activity showing a greater fluorescence potential than cells that did not undergo an upregulation of serine protease activity. The green FAM reporter dye is excited at 488 nm and emits in the green wavelength range of 525 nm.

FLISP probes are cell permeant and non-cytotoxic at the concentrations suggested in the assay protocol. The FLISP FAM-Leu-CMK (FLCK) chymotrypsin enzyme detection assay kits can be used in conjunction with existing apoptosis detection protocols. They have been used successfully in tandem with the red SR FLICA caspase detection assay kits to show a parallel upregulation of chymotrypsin-like enzyme activity and caspase activation. Each kit includes the FLISP FLCK reagent, 10x wash buffer for removing excess FLISP probe following the FLISP incubation step, 10X fixative to stabilize cells if next day analysis is preferred, Propidium Iodide vital stain for necrotic cell detection, and Hoechst 33342 dye to visualize nuclear morphology. FLISP FLCK-stained cells may be analyzed using flow cytometry, fluorescence microscopy, and fluorescence plate reader evaluation methodology.