| Solution Information | help | |
| Enzyme: | ADAM17 | |
| inhibitor: | BDBM41667 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay Overview: The purpose of this assay is to determine the selectivity of compounds that were identified as hits against ADAM10 during a previous set of experiments entitled "QFRET-based biochemical primary high throughput screening assay to identify exosite inhibitors of ADAM10" (AID 720582). 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 nominal 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 dispensed into 1536 microtiter plates. Compounds are added to plate (final | |
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