SR-101-Leu-CMK Red FLISP Assay Kit | SR-101-Leu-CMK assay kit

SR-101-Leu-CMK Red FLISP Assay Kit

Cat. Num. AAA258018

• Synonyms: SR-101-Leu-CMK Red FLISP; SR-101-Leu-CMK Red FLISP Assay Kit; SR-101-Leu-CMK assay kit

• Reagents: SLCK
• Sample Protocol: Culture cells up to 1 x 10^6 cells/mL. Follow your experimental protocol where chymotrypsin-like enzyme activity will be investigated; create positive and negative controls for comparing chymotrypsin activity. Reconstitute the vial of FLISP reagent with the appropriate volume of DMSO to form the stock concentrate (can be frozen for future use). Dilute the stock concentrate with PBS to form the 50X sample spiking solution. Add 6-10 uL of the FLISP spiking solution directly to a 300-500 uL aliquot of your cell culture for labeling. Incubate 30 minutes -1 hour at 37 degree C in the dark. Wash cells 3X with fresh media or 1x working dilution of FLISP wash buffer. If desired, label cells with Hoechst stain. If desired, fix cells for future analysis. Analyze data using a fluorescence microscope, plate reader, or flow cytometer.
• 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
• Preparation and Storage: FLISP at -20 degree C; Other Kit Components at 2 - 8 degree C

Testing Data

( Suspension cells were treated with an agent toinduce serine protease activity, (bottom), orDMSO, a negative control (top), then incubatedwith ICT's red serine protease inhibitor probe,SR-FLISP, washed, and analyzed with a flowcytometer. The experimental reatment inducedserine protease activity in 56.3% of theexperimental cells (M2, bottom right), whereasthe negative control treatment exhibited serineprotease activity in only 13.5% of the cellpopulation (M2, top right). This is a ratio of 4:1.(Dr. Brian W. Lee & Sally Hed, ICT).)

Testing Data #2

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Related Product Information for SR-101-Leu-CMK assay kit

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. This kit utilizes the probe SLCK, which contains a leucine (L) chymotrypsin-targeting amino acid residue linked on the amino terminus with a sulforhodamine 101 (SR) 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 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). 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. The red fluorescent SLCK FLISP probe is 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-L-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-L-CMK reagent, 10X wash buffer, 10X fixative, and Hoechst 33342 dye. SR101-L-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.

Background: Chymotrypsin-like enzymes cleave their substrate proteins at the carboxy terminal end 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 SLCK FLISP probe, consisting of SR101-Leu-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 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 [5].

Product Categories/Family for SR-101-Leu-CMK assay kit

Apoptosis Assays/Serine Protease Detection

References

Czapinska, H. and J. Otlewski. 1999. Structural and energetic determinants of the S1-site specificity in serine proteases. Eur. J. Biochem. 260: 571-595. Grabarek, J., M. Dragan, B.W. Lee, G.L. Johnson, and Z. Darzynkiewicz. 2002. Activation of chymotrypsin-like serine proteases during apoptosis detected by affinity-labeling of the enzymatic center with fluoresceinated inhibitor. Int. J. Oncol. 20: 225-233. Zhu, H., D. Dinsdale, E.S. Alnemri, and G.M. Cohen. 1997. Apoptosis in human monocytic THP-1 cells involves several distinct targets of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK). Cell Death Diff. 4: 590-599. Grabarek, J., L. Du, G.L. Johnson, B.W. Lee, D. J. Phelps, and Z. Darzynkiewicz. 2002. Sequential activation of caspases and serine proteases (Serpases). Cell Cycle 1: 124-131. Grabarek, J., M. Dragan, B.W. Lee, G.L. Johnson, and Z. Darzynkiewicz. 2002. Activation of chymotrypsin-like serine protease(s) during apoptosis detected by affinity-labeling of the enzymatic center with fluoresceinated inhibitor. Int. J. Oncol. 20: 225-233.