Go Back FAM-Phe-DAP FLISP Assay Kit--FFDAP

Product Detail

Detect changes in intracellular chymotrypsin-like enzyme activity in whole living cells with the FLISP, Fluorescent-Labeled Inhibitors of Serine Proteases, product line of assay kits. These kits utilize either a leucine (L) or phenylalanine (F) chymotrypsin-targeting amino acid residue linked on the amino terminus with either a carboxyfluorescein (FAM) or sulforhodamine 101 (SR) fluorescent reporter tag. The carboxyl end of the F or L amino acid residue contains a reactive group that targets the catalytic site of serine proteases, consisting of either a chloromethyl ketone (CMK) or (aminoalkyl) phosphonate diphenyl ester (DAP) reactive group.

After quickly penetrating the lipid bilayer membranes of the target cell population, the chymotrypsin-targeting FLISP probes will interact with the active catalytic sites of chymotrypsin-like proteases, quickly forming covalent bonds with either the reactive site histidine (N-H) (with the CMK-type FLISP probes) or the reactive serine OH group (when DAP-containing FLISP probes are utilized). In either case, unbound FLISP reagent is easily removed during the wash step, leaving cells with greater quantities of active chymotrypsin-like enzyme activity with a greater fluorescence potential than cells that did not undergo an upregulation of serine protease activity. FLISP probes bearing FAM reporter dyes are excited at 488 nm and emit in the green wavelength range of 525 nm. The red fluorescence FLISP probes that utilize sulforhodamine 101 as the reporter dye are excited at 590 nm and emit at 620 nm.

FLISP probes are cell permeant and non-cytotoxic at the concentrations suggested in the assay protocol. The FAM-F-DAP chymotrypsin enzyme detection assay kits can be used in conjunction with your existing apoptosis detection protocols. They can be used successfully in tandem with the Red FLICA apoptosis detection assay kits to show a parallel upregulation of chymotrypsin-like enzyme activity and caspase activation. Each kit includes either 25 tests or 100 tests of FAM-F-DAP reagent, 10X wash buffer for removing excess FLISP probe following the FLISP incubation step, 10X fixative to stabilize cells if needed for next day analysis, PI vital stain for necrotic cell detection, and Hoechst 33342 dye to stain apoptotic nuclei. FAM-F-DAP stained cells can be analyzed using flow cytometry, fluorescence microscopy, and fluorescence plate reader evaluation methodology.