|
Comment
|
Biotinylated peptide-fluoromethyl ketone inhibitor of Caspases-3/CPP32, -7, -1, -4, -8, and -10. The CH2F (fluoromethyl ketone) inhibitor has several advantages over other types of derivatives: penetrates cell membranes, not toxic to cells, irreversible inhibition.
Caspase-3/CPP32 is a member of the cysteine proteases family involved in apoptosis induction. All apoptotic pathways studied to date involve proteolytic activation of Caspase-3/CPP32 as a central event in the progression of cell death. Although the death-inducing consequences of Caspase-3/CPP32 activation have not been conclusively established, several crucial substrates for the protease have been identified in vitro, including DNA-dependent protein kinase, Poly(ADP-ribose) Polymerase (PARP),Replication factor C, and Gelsolin. These substrates are involved in the later stages of apoptosis, strongly suggesting that Caspase-3/CPP32 has a key role in promoting the final processes leading to cell death.
Binds to and inhibits Caspases-3/CPP32, -7, -1,-4, -8, and -10. Strong inhibition of Caspase-3 and Caspase-7 but no inhibition of Caspase-2. Moderate inhibition of Caspase-1 and Caspase-4. Very weak inhibition of Caspase-6.
Dissolve Caspase-3/CPP32 Inhibitor/Biotin Tag at 20 mM
in DMSO (high purity 99.9%) before use.
Method:
1. Grow and treat 1 x 10 6 cells at the appropriate dose and time to obtain 50-60% apoptosis.
2. Collect cells by centrifugation, remove supernatant and re-suspend cell pellet in an initial volume of 1/1,000th of the original media volume (10 μL for 10 mL media).
3. Add 20 μL of 2X Biotin-DEVD-FMK in MGD buffer (Final Biotin-DEVD-FMK concentration = 20 μM.)
MGD buffer:
50 mM NaCl
2 mM MgCl2
5 mM EDTA
10 mM HEPES 7.0
1 mM DTT Protease Inhibitor
4. Freeze/thaw 3X to lyse cells.
5. Incubate at 37°C for 15 minutes. Spin two minutes at 14K to remove cell debris.
6. Transfer supernatant to new tube, add 13 μL of 4X SDS-PAGE buffer and run all on 10% SDS-PAGE gel and transfer to PVDF membrane.
7. Block one hour and incubate with streptavidin-HRP (1/1,000) four hours. Develop by ECL. NOTE: The Carboxyl groups are OMe ester form to enhance cell permeability. If purified enzyme is used, esterase should be added for the hydrolysis of the ester groups.
For extended use in vivo or in vitro:
For experiments extending 12 to 48 hours, fresh inhibitor may have to be added (injected) due to inactivation of the inhibitor by endogenous cysteine proteases.
|
