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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.
|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|
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.
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