| Assay Method Information | |
| | MALT1 Inhibition Assay |
| Description: | The standard reagent formulations used in this assay were prepared and stored as follows. A 1.1 M solution of sodium citrate (161.3 g, 500 mL) was prepared and stored at room temperature. A 10% (w/v) CHAPS solution (2.0 g, 20 mL) was prepared and stored at 4 C. A 500 mM HEPES solution having pH of 6.89 was prepared from 200 mL of a 1 M HEPES solution having a pH of 7.5 using concentrated hydrochloric acid and brought to a final volume of 400 mL with MILLI-Q H2O. The substrate, 10 mM Ac-LRSR-AMC Peptide, was prepared (10 mg, 1.370 mL DMSO) and stored at −20 C.[1801]Compounds were plated to provide a 2% DMSO final concentration using a ProxiPlate-384 Plus F Black 384-shallow well microplate. The assay-ready plates were equilibrated to room temperature. A reaction buffer (30 mL total volume) was prepared by combining HEPES (pH 6.89, 25 mM, 1.5 mL), sodium citrate (660 mM, 18.0 mL), T-CEP (1 mM, 0.06 mL), EDTA (0.1 mM, 0.06 mL), CHAPS (0.05%, 0.15 mL), DMSO (2%, 0.6 mL), and MILLI-Q H2O (10.23 mL; 9.63 mL when backfilled to 2% DMSO) followed by thorough mixing. DMSO was added only when compound plates had not been DMSO-backfilled to 2%.[1802]MALT-1 was thawed and kept on ice. Peptide substrate was thawed on the bench under ambient conditions. A MALT-1 enzyme working stock was prepared from Avi-tagged FL MALT-1 (40 nM in a prepared reagent volume of 16.5 μL) and the reaction buffer (13.0 mL). MALT1 working stock (5 μL) was added to each well of the microplate. MALT-1 was pre-incubated with compounds for 30 minutes at room temperature.Two substrate working stocks (Km and 10 Km) were prepared. The 1 Km substrate was prepared from Ac-LRSR-AMC Peptide (50 μM in a prepared reagent volume of 35.0 μL) and the reaction buffer (6.965 mL). The 10 Km substrate was prepared from Ac-LRSR-AMC Peptide (280 μM in a prepared reagent volume of 196.0 μL) and the reaction buffer (6.804 mL). The reaction was initiated by the addition of substrate working stock (5 μL, 50 μM) to Km plates and the addition of substrate working stock (5 μL 280 μM) to 10 Km plates. The plates were covered and incubated on the bench at room temperature for 90 minutes.Fluorescence intensity was determined using a CLARIOstar microplate reader (BMG LABTECH) using the optimised AMC mode. Before taking readings, a 70% gain was applied on the neutral control well. |
| Affinity data for this assay | |
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