| Assay Method Information | |
| | MALT-1 Inhibition Assay |
| Description: | MALT-1 paracaspase activity was measured using the fluorogenic substrate Ac-LRSR-Rh110-DP (purchased from Biosantan GmbH). Proteolytic cleavage of the peptide—rhodamine conjugate results in an increase of rhodamine fluorescence which is inhibited by test compounds. Test compounds were diluted in DMSO in a series of 10 semi-log step doses, 15 nL of each compound dose were dispensed in 384 well polypropylene plates (HiBase non-binding, Greiner Bio-One cat #784900). All other assay components were diluted to appropriate working concentrations in assay buffer composed of: 200 mM Tris-HCl (pH 7.5; Sigma-Aldrich cat #T2663-1L), 0.1 mM EGTA (Sigma-Aldrich cat #E3889-10G), 0.05% CHAPS—Sigma-Aldrich cat #C9426-1G), 1 mM TCEP (Sigma-Aldrich cat #646547-10×1 mL), 0.8 M sodium citrate (Sigma-Aldrich cat #S1804-500G). Recombinant human MALT-1 (amino acids 340-824, accession NP_006776.1) was added to compound doses and equilibrated for 40 minutes at rt. The reaction was initiated by addition of substrate. Final concentrations of MALT-1 and substrate were 3 nM and 10 μM respectively. Reactions were incubated in the dark for 60 minutes at 25° C. Fluorescence was measured in a PHERAstar FSX plate reader (BMG LABTECH) with optical setup for excitation at 485 nM and emission at 520 nM, focal height of 11.8 mm, 20 flashes, gain 300. Percent inhibition values were calculated from relative fluorescence units at different doses and fitted to a 4-parameter logistic curve to determine IC50 values. |
| Affinity data for this assay | |
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