This Caspase-8 Fluorometric Assay Kit is designed to detect caspase-8 cleaving activity, Caspase-8 (FLICE/MACH-1) is a 55 kDa cytosolic protein with homology to the CD95/Fas-associated signal transduction molecule, FADD, in addition to its homology with other caspases. The kit contains a caspase-8 fluorogenic substrate (Ac-IETD-AFC), a caspase-8 aldehyde inhibitor (Ac-IETD-CHO) and assay buffers. Cells that are suspected or have been induced to undergo apoptosis are first lysed to collect their intracellular contents. The cell lysate can then be tested for protease activity by the addition of a caspase-specific peptide that is conjugated to the fluorescent reporter molecule 7-amino-4-trifluoromethyl coumarin (AFC). The cleavage of the peptide by the caspase releases the fluorochrome that, when excited by light at 400 nm wavelength, emits fluorescence at 505 nm. The level of caspase enzymatic activity in the cell lysate is directly proportional to the fluorescence signal detected with a fluorimeter or a fluorescent microplate reader.
|Detection Method||Fluorescence (400 nm excitation filter and 505 nm emission filter)|
|Applications||Detect early/middle stages of apoptosis; differentiate apoptosis from necrosis.|
|Features & Benefits||• Simple one-step procedure; takes 1-2 hours
• Fast and convenient
• Comparison of the fluorescence of AFC from an apoptotic sample with an uninduced control allows determination of the fold increase in FLICE activity.
|Kit Components||• Cell Lysis Buffer
• 2X Reaction Buffer
• IETD-AFC (1mM)
• DTT (1 M)
20tests ; 50tests ; 100tests
Store at ≤ -20 °C for up to 6 months after initial use. Avoid repeated freeze-thaw cycles.
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