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Serine proteases belong to a multigene family of proteins that are involved in the post-translational processing of many polypeptides. They have central roles in regulating a wide range of physiological processes, including digestion, coagulation, malignancy, inflammation, fertilization, and development. Serine protease kits are research tools for measuring chymotrypsin-like serine protease activation. The serine protease inhibition methodology is based on the use of a fluorochrome-labeled, cell-permeable inhibitor of chymotrypsin-like serine proteases. The inhibitors in the serine protease kits are fluorochrome-labeled analogs of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). TPCK is an inhibitor that covalently binds to the active centers of chymotrypsin-like enzymes. The protease inhibitor in the kits is either carboxyfluorescein (FFCK), used to measure green fluorescence or Sulforhodamine 101 [also know at Texas-Red™ (TRFCK)], used to measure red fluorescence.
Note: the FFCK and TRFCK terminology represents the current nomenclature of “F” for phenylalanine as opposed to “P” which was in when the TPCK inhibitor was originally described.
FFCK inhibitor and TRFCK inhibitor are designed to be used with living cells and do not require any cellular fixation or permeabilization steps. The nomenclature “serpase” was first defined in analogy to caspases to describe members of the serine protease family that are activated during apoptosis. However, compared to caspases, much less information is known about apoptosis-associated serpases. IMGENEX's serine protease (SerPase™) kits are useful research tools for helping to elucidate the roles of a serine protease in apoptosis, cell survival, and other signal transduction pathways.
When added to a population of cells, the fluorescently labeled inhibitor [FFCK or TRFCK] diffuses into the cells and covalently binds to active sites of proteases. Bound inhibitor is retained within the cell, and unbound inhibitor that diffuses out is washed away. The remaining fluorescent signal is a measure of the active proteases that were present in the cell at the time the FFCK or TRFCK inhibitor was added. |
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Analysis by flow cytometry and fluorescence microscopy using control and staurosporine treatment (1µM/16 h). HeLa cells were labeled with serine protease inhibitor TRFCK for analysis by flow cytometry or with both serine protease inhibitor TRFCK (red) and Hoechst DNA counterstain (blue) for analysis by fluorescence microscopy. Control: top micrographs and unshaded histogram. Staurosporine-treated: bottom micrographs and shaded histogram in red.
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