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| | In Vitro Protease Cleavage Assay |
| Description: | To screen for small molecular weight compounds that can inhibit MALT1 protease activity, recombinant GSTMALT1 was purified from E. coli to establish an in vitro protease cleavage assay suitable for high throughput screening (HTS). GSTMALT1 was incubated for 1 h at 30° C. in the presence of 50 uM of the tetrapeptide substrate Ac-LRSR-AMC, which is derived from the MALT1 cleavage site in the C-terminus of BCL10.7 Proteolytic activity was determined by measuring the increase of fluorescence, which is emitted after cleavage and the accompanying release of the fluorophore AMC (FIGS. 1A and B). MALT1 catalyzed cleavage of Ac-LRSR-AMC is evident from a robust increase in fluorescence intensity over time. Mutation of the conserved cysteine (C453A) in the paracaspase domain of MALT1 (Isoform B) completely abolished MALT1 catalytic activity (FIG. 1A). Similar to arginine-lysine specific metacaspases, the MALT1 protease has a high preference for cleaving after an arginine residue. Consistent with this Z-VRPR-FMK, which was initially designed as a metacaspase antagonistic peptide, also completely blocked MALT1 cleavage activity at low nanomolar concentrations, emphasizing the high similarity of the paracaspase to plant metacaspases. |
| Affinity data for this assay | |
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