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Caspase 8 Assay Kit (colorimetric) (ab39700) is a simple and convenient assay to quantify the activity of caspase 8 in cell lysates, based on the recognition of the sequence Ile-Glu-Thr-Asp (IETD). The assay is based on spectrophotometric detection of the chromophore p-nitroanilide (pNA) after it is cleaved from the labeled substrate IETD-pNA. The pNA light emission can be quantified using a spectrophotometer or a microtiter plate reader at OD 400 – 405 nm. Comparison of the absorbance of pNA from an apoptotic sample with an un-induced control allows determination of the fold increase in Caspase 8 activity.
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The caspase family of highly conserved cysteine proteases play an essential role in programmed cell death (including apoptosis, pyropoptosis and necroptosis).
Mammalian caspases can be subdivided into three functional groups: initiator caspases (Caspase 2, 8, 9 and 10), executioner caspases (Caspase 3, 6 and 7), and inflammatory caspases (Caspase 1, 4, 5, 11 and 12). Initially synthesized as inactive pro-caspases, caspases become rapidly cleaved and activated in response to granzyme B, death receptors and apoptosome stimuli. Caspases will then cleave a range of substrates, including downstream caspases, nuclear proteins, plasma membrane proteins and mitochondrial proteins, ultimately leading to cell death.
Caspase 8 (CASP8/FLICE, EC:188.8.131.52) is the most upstream caspase involved in the activation of apoptosis through the extrinsic pathway, mediated by CD95 (Fas) receptor and TNFR. Caspase 8 is recruited to the receptors through the adapter molecule FADD, resulting in the formation of the aggregate called death-inducing signaling complex (DISC) and proteolytic activation of caspase 8. The active dimeric enzyme is then liberated from the DISC and free to activate downstream apoptotic proteases. Inhibition or inactivation of caspase 8 is required for induction of necroptosis.
|2X Reaction Buffer||4 x 2ml|
|Cell Lysis Buffer||1 x 100ml|
|Dilution Buffer||1 x 100ml|
|DTT||1 x 400µl|
|IETD-pNA||1 x 500µl|
Active caspase 8 in control (CTRL) Jurkat cells (10e6/mL) or in cells after four hours exposure to 50 ng/mL anti-Fas Ab (αFas) (MBL), or pretreated one hour with 50 μM Z-VAD(OMe)-FMK (ab120487) followed by four hours with αFas. Background signal subtracted, duplicates; +/- SD.
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