Description
Description: Detect active caspase-2 with the FAM FLICA Caspase 2 Assay Kit. This in vitro assay employs the green fluorescent inhibitor probe FAM-VDVAD-FMK to label active caspase-2 enzymes in living cells or tissue samples. Analyze the fluorescent signal using fluorescence microscopy, a fluorescent plate reader, or by flow cytometry. FLICA (Fluorescent Labeled Inhibitor of Caspases) caspase detection probes are comprised of an affinity peptide sequence, a fluoromethyl ketone (FMK) moiety that facilitates an irreversible binding event with the active caspase enzyme, and a fluorescent tag (either carboxyfluorescein or sulforhodamine B) reporter. For a caspase-2 inhibitor, the enzyme recognition sequence is valine-aspartic acid-valine-alanine-aspartic acid (VDVAD). The FLICA probe interacts with the enzymatic reactive center of an activated caspase via the recognition sequence, forming a covalent thioether adduct with the enzyme through the FMK moiety and inhibiting further enzymatic activity. FLICA probes are cell permeant and non-cytotoxic. Unbound FAM- FLICA reagent is washed away; the remaining green fluorescent signal is a direct measure of caspase-2 activity at the time the probe was added. Detection of nuclear morphology is also possible with the additional kit components Hoechst 33342 and Propidium Iodide.
Background: Apoptosis is an evolutionarily conserved form of cell suicide mediated by a cascade of proteolytic enzymes called caspases. Pro-apoptotic signals activate the enzymatic cascade resulting in the cleavage of protein substrates, leading to the disassembly of the cell (1-4). Caspases have been identified in organisms ranging from C. elegans to humans. Members of the mammalian caspase family of cysteinyl aspartate-specific proteases play distinct roles in apoptosis and inflammation. There are two types of caspases; the initiators (caspases 8, 9, and 10) and the effector caspases (caspases 1, 2, 3, 4, 6, 7, 12, and 13). The initiator caspases 8 and 10 are also referred to as the extrinsic apoptosis pathway that originates upon activation of cell surface death receptors. Caspases 8 and 10 are monomers that bind to death receptor proteins through their death effector domain (DED) structure. Caspase 9 is also called the intrinsic pathway that results from the mitochondrial release of cytochrome c. The initiator caspase 9 monomer binds other proteins through their caspase activation and recruitment domain (CARD). The initiator caspase -protein interaction results in dimerization of the initiator caspases that leads to their activation. These activated initiator caspases then cleave the effector pro-caspases at specific aspartic acid residues to yield large (20 kDa) and small (10 kDa) subunits that then assemble into the heterotetrameric, catalytically active form of the caspase effector enzymes (5, 6). Active caspase enzymes exhibit catalytic and substrate specificities comprised of short tetra-peptide amino acid sequences that must contain an aspartate in the P1 position (7 - 9). These preferred tetra- peptide sequences have been used to derive peptides that specifically compete for caspase binding (4 - 6). In addition to the distinctive aspartate cleavage site at P1, the catalytic domains of the caspases require typically four amino acids to the left of the cleavage site with P4 as the prominent specificity-determining residue (9). In contrast to this tetrapeptide specificity, the tri-peptide VAD is able to bind to the active site of every caspase family member studied. Furthermore, addition of a fluoromethyl ketone (FMK) to the tri-peptide results in an irreversible linkage and permanent inactivation of the cysteine protease enzyme (10). Accordingly, the Z-VAD-FMK inhibitor has been shown in numerous studies to effectively inhibit the induction of apoptosis by blocking caspase activation (9, 11). Furthermore, substitution of the amino terminal benzyloxycarbonyl blocking group (Z-) with a detection moiety, such as a fluorescent dye, yields a probe that allows for the detection of caspase activity (12 - 14). FLICA: FLuorescent-Labeled Inhibitors of Caspases The FLICA methodology of caspase detection is available in kit form for assessing individual or poly-caspase activity in cultured cells and tissues. The non-toxic, cell-permeant FLICA reagent enters each cell, where it will irreversibly bind to activated caspases with a preference for its target peptide sequence. For example, active caspase-2 has a high affinity for the peptide sequence V-D-V-A-D. Because the FLICA reagent becomes covalently coupled to the active enzyme, it is retained within the cell during wash steps, while any unbound FLICA reagent diffuses out of the cell and is washed away. The remaining green fluorescent signal is a direct measure of the amount of caspase activity present in the cell at the time the reagent was added. Cells that contain the bound FLICA can be analyzed by 96-well-plate based fluorometry, fluorescence microscopy, or flow cytometry. The carboxyfluorescein (FAM) FLICA reagent has an optimal excitation range from 490 - 495 nm, and emission range from 515 - 525 nm. Cells labeled with the FLICA reagent may be read immediately or preserved for 24 hours using the fixative. Unfixed samples may be subsequently analyzed with propidium iodide or Hoechst stain to detect changes in necrosis or nuclear morphology respectively. Other FLICA Caspase Detection Kits, containing the preferred caspase recognition amino acid sequences for poly caspases or caspase 1, 3, 6, 8, 9, 10, and 13, are also available with green or red fluorescence. Browse our FLICA page to learn more. Slee, E. A., C. Adrain, and S. J. Maritin. (1999) Serial Killers: ordering caspase activation events in apoptosis. Cell Death Differ. 6:1067-1074. Earnshaw, W.C., Martins, L.M., and Kaufmann, S.H. (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Ann. Rev. Biochem. 68:383-424. Hengartner, M.O. (2000) The biochemistry of apoptosis. Nature 407:770-816. Degterev, A., Boyce, M., and Yuan, J. (2003) A decade of caspases. Oncogene 22:8543-8567. Nicholson, D.W. (1999) Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ. 6:1028-1042. Thornberry, N.A., and Lazebnik, Y. (1998) Caspases: enemies within. Science 281:1312-1316. Cryns, V., and Yuan, J. (1998) Proteases to die for. Genes Dev. 12:1551 - 1570. Talanian, R.V., Quinlan, C., Trautz, S., Hackett, M.C., Mankovich, J.A., Banach, D., Ghayur, T., Brady, K.D., and Wong, W.W. (1997) Substrate specificities of caspase family proteases. J. Biol. Chem. 272:9677 - 9682. Garcia-Calvo, M., Peterson, E.P., Leiting, B., Ruel, R., Nicholson, D.W., and Thornberry, N.A. (1998) Inhibition of human caspases by peptide-based macromolecular inhibitors. J. Biol. Chem. 273:32608 - 32613. Rauber, P., Angliker, H., Walker, B., and Shaw, E. (1986) The synthesis of peptidylfluoromethanes and their properties as inhibitors of serine proteases and cysteine proteinases. Biochem. J. 239:633-640. Ekert, P.G., Silke, J., and Vaux, D.L. (1999) Caspase inhibitors. Cell Death Differ. 6:1081-1086. Bedner, E., Smolewski, P., Amstas, P., and Darzynkiewicz, Z. (2000) Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp. Cell Res. 259:308-313. Amstad, P.A., Yu, G., Johnson, G.L., Lee, B.W., Dhawan, S., and Phelps, D.J. (2001) Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors. BioTechniques 31:608-610. Smolewski, P., Bedner, E., Du, L., Hsieh, T.C., Wu, J.M., Phelps, D.J., and Darzynkiewicz, Z. (2001) Detection of caspase activation by fluorochrome- labeled inhibitors: multiparameter analysis by laser scanning cytometry. Cytometry 44:73-82