| Solution Information | help | |
| Enzyme: | Insulin-degrading enzyme | |
| inhibitor: | BDBM75806 | |
| 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 confirm activity of compounds identified as active in a set of previous experiments entitled, "Fluorescence polarization-based cell-based high throughput primary assay to identify inhibitors of insulin-degrading enzyme (IDE)" (AID 434962). The assay determines the ability of test compounds to inhibit endogenous cellular IDE activity by measuring shifts in the proportion of intact and cleaved forms of the IDE substrate Abeta. This fluorescence polarization (FP)-based assay (24) employs a derivatized Abeta- peptide [Fluorescein-Abeta-(1-40)-Lys-Biotin (FAbetaB)]. Cleavage of FAbetaB by IDE (provided by live HEK cells in the reaction) separates the Fluorescein moiety from the biotinylated moiety. Subsequent addition of avidin to the reaction increases the effective molecular weight of intact, biotinylated FAbetaB substrate, slowing their rotation rate and reducing the degree of depolarization of plane polarized light. In contrast, cleaved | |
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