Application Notes
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Inhibition of caspase-3, -7, -1, -4, -8, and -10 activities. For caspase-3 fluorometric assays using the Caspase-3 Fluorogenic Substrate (Cat. No. AC-003), Caspase-3(CPP32) Inhibitor can be used to assess the contribution of contaminating proteases to the overall rate of proteolysis.
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Comment
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Irreversible and cell permeable inhibitor of caspase-3 (CPP32, Apopain), a member of the CIE/CED-3 family of cysteine proteases. The CH2F (fluoromethyl ketone) inhibitor has several advantages over other types of derivatives: Penetrates cell membranes, is nontoxic to cells, irreversible inhibition. Caspase-3 is a member of the cysteine proteases family involved in apoptosis induction. All apoptotic pathways studied to date involve proteolytic activation of caspase-3 as a central event in the progression of cell death. Although the death-inducing consequences of caspase-3 activation have not been conclusively established, several crucial substrates for the protease have been identified in vitro, including DNA-dependent protein kinase, Poly(ADPribose) Polymerase (PARP), Replication factor C, and Gelsolin. These substrates are involved in the later stages of apoptosis, strongly suggesting that caspase-3 has a key role in promoting the final processes leading to cell death. Inhibition of caspase-3, -7, -1, -4, -8, and -10 activities. For caspase-3 fluorometric assays using the Caspase-3 Fluorogenic Substrate (ABIN924940), Caspase-3(CPP32) Inhibitor can be used to assess the contribution of contaminating proteases to the overall rate of proteolysis. Dissolve the Caspase-3(CPP32) Inhibitor in high purity DMSO (>99.9%) before use to make a stock solution of 20 mM.
For use on intact cells: 1. Prepare desired concentrated stock solutions as follows: 5 mg Z-DEVD-FMK in 37μl DMSO = 20 mM in 75μl DMSO = 10 mM in 1,50μl DMSO = 5 mM, etc.
2. Adding 2 μL of the above stock solutions to 1 mL of culture medium containing cells gives a fμl DMSO concentration of 0.2%. Adding 2 μL of a 10 mM stock solution to 1 mL of culture medium gives a final Z-DEVD-FMK concentration of 20 μM.
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