SR101-Phe-CMK Serine Protease Assay Kit

Product Discontinued

SR101-Phe-CMK Serine Protease Assay Kit Summary

• Description: 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.
• Specificity: Detect changes in intracellular chymotrypsin-like enzyme activity in whole living cells with the FLISP (TM), 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 (TM), 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 SR101-F-CMK FLISP chymotrypsin enzyme detection assay kits can be used in conjunction with your existing apoptosis detection protocols. Each kit includes either 25 tests or 100 tests of SR101-F-CMK 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, and Hoechst 33342 dye to stain apoptotic nuclei. SR101-F-CMK stained cells can be analyzed using fluorescence microscopy, and fluorescence plate reader evaluation methodology. If a green laser is available on your flow cytometer, this method may be utilized as well.
• Preparation Method: Target: chymotrypsin-like enzymes Excitation / Emission: 565 nm / 590 nm Method of Analysis: Flow Cytometer, Fluorescence Microscope, Fluorescence Plate Reader Types of Samples: cell culture, tissue
• Kit Type: Assay Kit

Applications/Dilutions

• Dilutions:
  • Flow Cytometry

Packaging, Storage & Formulations

• Storage: Storage of components varies. See protocol for specific instructions.

Alternate Names for SR101-Phe-CMK Serine Protease Assay Kit

• FLISP SR101-Phe-CMK Serine Protease
• Phe-CMK Serine Protease
• TR-Phe-CMK (TRFCK)

Background

Chymotrypsin-like enzymes cleave their substrate proteins at the carboxy terminus of amino acids containing hydrophobic aliphatic or aromatic R-group side chains such as those found on leucine and phenylalanine amino acid structures [1]. The red phenylalanine FLISP probe, consisting of SR101-Phe-CMK, is simply a fluorescent-labeled analog of the early chymotrypsin-like enzyme inhibitor N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) [2]. Early studies using topoisomerase inhibitors in HL-60 cells indicated that TPCK, N-tosyl-L-lysine chloromethyl ketone (TLCK) and other serine protease inhibitors were able to prevent internucleosomal DNA degradation associated with apoptosis [3]. Dual staining experiments have used our green FAM-F-CMK (chymotrypsin detection probe) and red sulforhodamine (SR)-VAD-FMK FLICA caspase detection probe in the presence or absence of z-VAD-FMK, TPCK, or TLCK caspase or serine protease inhibitors [4-5]. This work demonstrated a significant inhibitory effect of the caspase inhibitor, z-VAD-FMK, on chymotrypsin-associated FAM-F-CMK binding in cells. This group also showed that the chymotrypsin-like enzyme inhibitor, TPCK, had a strong suppressive effect on caspase activation and apoptosis induction. In contrast, the trypsin-like enzyme inhibitor, TLCK, had very little effect on caspase activation events as measured by the binding of the red FLICA probe SR-VAD-FMK in camptothecin-induced cell populations

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Kits are guaranteed for 6 months from date of receipt.