Caspase 8, cleaved, Human, BioAssay(TM) Cell-Based ELISA Kit (CASP8, Mch5)

Caspases are a family of cytosolic aspartate-specific cysteine proteases involved in the initiation and execution of apoptosis. They are expressed as latent zymogens and are activated by an autoproteolytic mechanism or by processing by other proteases (frequently other caspases). Human caspases can be subdivided into three functional groups: cytokine activation (caspase-1, -4, -5, and -13), apoptosis initiation (caspase-2, -8, -9, -and -10), and apoptosis execution (caspase-3, -6, and -7). Caspases are regulated by a variety of stimili, including APAF1, CFLAR/FLIP, NOL3/ARC, and members of the inhibitor of apoptosis (IAP) family such as BIRC1/NAIP, BIRC2/cIAP-1, BIRC3/cIAP-2, BIRC4/XIAP, BIRC5/Survivin, and BIRC7/Livin. IAP activity is modulated by DIABLO/SMAC or PRSS25/HTRA2/Omi. \n\nSample Type:\nWhole cells\n\nIntended Use:\nThis BioAssay(TM) Cell-Based ELISA contains the components required to run an ELISA using fluorogenic substrates to measure Cleaved Caspase-8 (Asp391) in whole cells. This simple and efficient assay eliminates the need to prepare cell lysates and can be used to investigate signaling pathways and the effects of inhibitors on cells.\n\nTest Principle:\nCells are grown in 96-well microplates and stimulated with ligands. Following stimulation, cells are fixed and permeabilized in the wells. The target protein amount is measured using a double immunoenzymatic labeling procedure. The cells are simultaneously incubated with two primary antibodies: an antibody specific for the target protein and a normalization antibody that is specific for GAPDH, a housekeeping protein. The primary antibodies are derived from different species. Two secondary antibodies recognizing the different species are labeled with either horseradish-peroxidase (HRP) or alkaline phosphatase (AP), and two spectrally distinct fluorogenic substrates for either HRP or AP are used for detection. The fluorescence of the target protein is normalized to that of the total GAPDH in each well for the correction of well-to-well variations. This two-wavelength assay results in precise analysis of Cleaved Caspase-8 (Asp391) with good reproducibility.\n\nKit Components:\nCleaved Caspase-8 (Asp391) Antibody, 1x vial \nTotal GAPDH (Ms) Antibody, 1x vial\nHRP-conjugated anti-rabbit IgG, 1x110ul\nAP-conjugated anti-mouse IgG, 1x110ul\nSubstrate F1 Concentrate, 1x50ul\nF1 Diluent, 1x10ml\nSubstrate F2, 1x10ml\nBlocking Buffer, 1x35ml\nWash Buffer, 5X concentrated, 1x60ml\nMicroplate, 1x96wells\nPlate Sealers, 4x adhesive strips\n\nStorage and Stability:\nStore powder at 4 degrees C liquid at -20 degrees C. Store other components at 4 degrees C. Stable for at least 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

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