Caspase 4 Human Caspase Cysteine Peptidase Enzymatic LeadHunter Assay - TW

Procedure Summary:

Human recombinant caspase 4 expressed in E. coli is used. Test compound and/or vehicle is preincubated with 10 U/ml enzyme‡ in modified HEPES buffer pH 7.4 for 15 minutes at 37⁰C. The reaction is initiated by addition of 25 μM Ac-LEVD-AFC for another 60 minute incubation period. Determination of the amount of AFC formed is read spectrofluorimetrically at 400 nm/510 nm. Compounds are screened at 10 μM.

Substrate:

25 µM Ac-LEVD-AFC

Incubation:

60 min at 37°C

Control Inhibitor:

Ac-LEVD-CHO

Criteria For Significance:

≥ 50% of max stimulation or inhibition

Test Concentration / Dose:

Suggested concentration for initial testing is 10 uM

Test Sample Requirements:

Minimum for 1) Screen: 25 μl of 10 mM stock -OR- 1 mg (pre-weighed). 2) Dose Response: 60 μl of 10 mM stock -OR- 1 mg (pre-weighed).

Minimum Order Quantity:

2