| Assay Method Information | |
| | MALT1 Protease Assay 2 (Assay 2) |
| Description: | MALT1 protease activity was assessed in vitro by measuring the cleavage of a fluorogenic tetrapeptide substrate.For a higher sensitivity assay, commercially available FL hMALT1 enzyme (Abcam, 1-10 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, 1 mM TCEP and 0.7 M sodium citrate.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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