| Assay Method Information | |
| | Enzyme Inhibition Assay |
| Description: | Compounds were dissolved in dimethylsulfoxide (DMSO), and further diluted in 0.1 M sodium acetate buffer (with 150 mM sodium chloride, pH 4.5). The solution with no compound was used as a negative control. Then, 100 L of these solutions (compound concentration: 3 uM), 1 L of recombinant human -secretase (rhBACE-1, R&D), and 5 uL of the fluorescent substrate peptide were mixed in a black 96-well plate (Nunc). After mixtures were incubated in the dark at 37 C. for 2 hr, the fluorescence intensities of the mixtures were measured by fluorescence microplate reader (Wallac) at 540 nm for excitation and at 590 nm for emission. The inhibition ratio was calculated as a percentage of the negative control. The sequence of the peptide was Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Lys-Arg, and labeled with fluorescent donor (Cy3) at Ser-1 and with quencher (Cy5Q) at Lys-9, respectively (Invitrogen). |
| Affinity data for this assay | |
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