| Assay Method Information | |
| | MALT1 Enzymatic Activity Inhibition |
| Description: | Compound activity was determined using recombinant MALT1 protein (Creative Biomart, Cat #MALT1-28H) and Ac-LRSR-AMC Substrate in an in vitro enzymatic reaction. The enzymatic assay used to determine activity was a Fluorescence assay using a Microplate Reader ClarioStar Plus. The enzymatic reaction was carried out in 1× Assay Buffer (50 mM HEPES pH 7.2-7.4, 100 mM NaCl, 900 mM Sodium citrate, 10 mM DTT). The compounds were dispensed on a 384-well Diamond Well Plate (Axygen, Cat #P-384-120SQ-C—S) using the Biomek FX liquid handling system at 100× solutions of compounds in DMSO. 2.5×MALT1 mix (final concentration 1.5 ng/μl of MALT1) was prepared in 1× Assay Buffer and 8 μl of mixture per well were added into 384w white Reaction Plates with NBS (Corning, Cat #4513). 8 μl of 1× Assay Buffer were used for negative control. Plates were centrifuged for 1 min at 200 g. Next, the compounds were added to Reaction plates using Biomek station via following steps: 1 μl of 100× compounds (in DMSO) was mixed thoroughly with 19 μl of 1× Assay Buffer, then 4 μl of this mixture were added to Reaction plates with 8 μl of MALT1 mix. Plates were centrifuged for 1 min at 200 g and incubated for 10 minutes at rt. Next, 2.5× Ac-LRSR-AMC mix (final concentration 1 μM of Ac-LRSR-AMC) was prepared in 1× Assay Buffer and 8 μl of mixture per well were added to Reaction Plates using Biomek station. Plates were centrifuged for 1 min at 200 g, then the fluorescence was measured immediately using Microplate Reader. Plates were incubated for 30 minutes at 37° C., then for 30 minutes at rt. The fluorescence was measured after the whole incubation using Microplate Reader. The reaction signal was calculated as the subtraction of the first data set values from the second. |
| Affinity data for this assay | |
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