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Serine proteases belong to a multigene family of proteins that are involved in the post-translational processing of many polypeptides. These proteases have central roles in regulating a wide range of physiological processes, including digestion, coagulation, malignancy, inflammation, fertilization, and development. SerPase™ Kits are research tools for measuring chymotrypsin-like serine protease activation. The serpase methodology is based on fluorochrome-labeled, cell permeable inhibitors of chymotrypsin-like serine proteases. The inhibitors in the SerPase™ 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 kits contain either carboxyfluorescein (FFCK) or Sulforhodamine 101[also know at Texas-Red™ (TRFCK)] labeled inhibitor to measure green or red fluorescence, respectively. [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 and TRFCK inhibitors 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 serine proteases that are activated during apoptosis. However, compared to caspases much less information is known about apoptosis-associated serpases. The SerPase™ Kits should be useful research tools for helping to elucidate the roles of serine proteases 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 of the cell 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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Cell analysis by flow cytometry and fluorescence microscopy. Control and staurosporine treated (1µM/16 h). HeLa cells were labeled with TRFCK for analysis by flow cytometry or with both TRFCK (red) and Hoechst DNA counterstain (blue) for analysis by fluorescence microscopy. Control cells: top micrographs and unshaded histogram. Staurosporine treated cells: bottom micrographs and shaded histogram in red.
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