| Assay Method Information | |
| | MALT1 Protease Assay 1 (Assay 1) |
| Description: | MALT1 protease activity was assessed in vitro by measuring the cleavage of a fluorogenic tetrapeptide substrate.[0581]A protein (MALT1-GS-Ub) comprising 6-His-hMALT-1 (residues 339-715), fused to ubiquitin with an 8× GGS linker, was expressed in E. coli, and purified using chitin resin to remove contaminants followed by affinity chromatography purification and size-exclusion chromatography, according to standard protocols.Briefly, MALT1-GS-Ub (300-600 nM) was incubated with substrate (Ac-LVSR-AMC, 100 μM) in reaction buffer comprising 50 mM HEPES (pH7.0), 25 mM KCl, 0.1% (v/v) CHAPS and 1 mM TCEP.Test compounds dissolved in DMSO were dispensed into assay plates (384-well, black, shallow ProxiPlates). 7 μl enzyme solution was added and incubated at room temperature for 30 minutes to allow compound binding to occur. 2 μl substrate solution was then added and the fluorescence (excitation 360 nm, emission 460 nm) read every 15 minutes using a suitable plate reader. Final assay DMSO concentration was 1%. Linearity over 60 minutes was confirmed, and assay signal was calculated by subtracting raw counts at time 0 from those at 60 minutes. % inhibition was calculated for each well, using no enzyme for 100% inhibition controls and no compound for 0% inhibition controls. IC50 values were calculated using GraphPad Prism using a 4-parameter non-linear regression curve fit. |
| Affinity data for this assay | |
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