| Solution Information | help | |
| Enzyme: | ADAM17 | |
| inhibitor: | BDBM31354 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay Overview: The purpose of this biochemical assay is to determine ADAM17 inhibitory dose response curves of compounds identified as active in a previous set experiments entitled "QFRET-based biochemical high throughput confirmation assay to identify exosite inhibitors of ADAM17". Specifically, this assay is used to identify compounds that inhibit ADAM17. This assay employs a fluorophore and quencher pair. F =EDANS fluorophore, Q = DABCYL quencher. When intact, EDANS emission at 460nm is quenched by DABCYL via fluorescence resonance energy transfer. Upon cleavage of the scissile bond (A~V) by ADAM protease, the distance between fluorophore and quencher increases resulting in fluorescence increase at 460nm. Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nomimal test concentration of 69.5 uM. Protocol Summary: Prior to the start of the assay, 2.5 microliters 2X ADAM17 enzyme (20 nM in Assay Buffer: 50 mM HEPES, 0.01% Brij, pH 7.5) are | |
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