Key features and details
- Assay type: Quantitative
- Detection method: Fluorescent
- Platform: Microplate reader
- Assay time: 1 hr 30 min
- Sample type: Adherent cells, Suspension cells
Product nameCaspase-9 Inhibitor Assay Kit
See all Caspase-9 kits
Sample typeAdherent cells, Suspension cells
Assay time1h 30m
Abcam's Caspase 9 Inhibitor Drug Detection Kit provides an effective means for screening caspase inhibitors using fluorometric methods. The assay utilizes synthetic peptide substrate LEHD-AFC (AFC, 7-amino-4-trifluoromethyl coumarin). Active caspase 9 cleaves the synthetic substrate to release free AFC which can then be quantified by fluorometry. Compounds to be screened can directly be added to the reaction and the level of inhibition of caspase 9 activity can be determined by comparison of the fluorescence intensity in samples with and without the testing inhibitors.
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Caspases have been shown to play a crucial role in apoptosis induced by various deleterious and physiologic stimuli. Inhibition of caspases can delay apoptosis, implicating a potential role in drug screening efforts.
Storage instructionsStore at -20°C. Please refer to protocols.
Components 100 tests 2X Reaction Buffer 1 x 10ml Active Caspase-9 1 x 100 units Caspase Inhibitor, Z-VAD-FMK (2 mM) 1 x 10µl Caspase Substrate LEHD-AFC (1 mM) 1 x 0.5ml DTT (1 M) 1 x 100µl
RelevanceCaspases are cysteine proteases, expressed as inactive precursors, that mediate apoptosis by proteolysis of specific substrates. Caspases have the ability to cleave after aspartic acid residues. There are two classes of caspases involved in apoptosis; initiators (activation by receptor cluster) and effectors (activation by mitochondrial permeability transition). Proapoptotic signals autocatalytically activate initiator caspases, such as Caspase 8 and Caspase 9. Activated initiator caspases then process effector caspases, such as Caspase 3 and Caspase 7, which in turn cause cell collapse. Caspase 9 (also known as ICE like apoptotic protease 6 (ICE LAP6), apoptotic protease Mch6, and apoptotic protease activating factor 3 (Apaf3)) is a member of the peptidase family C14 that contains a CARD domain. It is active as a heterotetramer and has been reported to have two isoforms. ProCaspase 9 is approximately 47 kD. It is present in the cytosol and, upon activation, translocates to the mitochondria. Caspase 9 is involved in the caspase activation cascade responsible for apoptosis execution and cleaves/activates Caspase 3 and Caspase 6. It becomes activated when recruited to the Apaf1/cytochrome c complex.
- Caspase 9
Titration of the caspase 9 inhibitor Z-LEHD-FMK (ab142026) (duplicates; +/- SD).
Titration of caspase inhibitor Z-VAD-FMK (ab102497) (duplicates; +/- SD).
Caspase activity (RFU) in presence of 0µM - 40µM of z-VAD-FMK (generic caspase inhibitor), assessed using LEHD-AFC as caspase 9 substrate and following Caspase 9 Inhibitor Drug Detection Kit (ab102493) protocol. Lanes:
1.- Background Control: no Caspase 9, no z-VMAD-FMK
2.- Positive Inhibition Control: no Caspase 9, +z-VAD-FMK
3.- Positive Control: + Caspase 9, no z-VAD-FMK
4.- Caspase 9 + 2.5µM z-VAD-FMK
5.- Caspase 9 + 10µM z-VAD-FMK
6.- Caspase 9 + 40µM z-VAD-FMK
ab102497 has not yet been referenced specifically in any publications.