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Assay Method Information |
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| | Fluorescence Quench Assay |
| Description: | Inhibitory activity of compounds was assessed by a fluorescence quench assay of BACE activity using commercially available substrate HiLyte Fluor.TM.488-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys-(QXL.TM. 520)-OH (AnaSpec, San Jose, Calif.) and truncated human beta-secretase (residues 1-458, His.sub.6-tagged at the C-terminus) expressed in insect cells D. melanogaster S2 using a baculovirus expression system (Mallender et al., Characterization of recombinant, soluble beta-secretase from an insect cell expression system., Mol Pharmacol 59:619-26, 2001). The assay was performed at room temperature in 96-well white opaque Optiplates aque Optiplates (PerkinElmer, Waltham, Mass.) in a total volume of 200 .mu.l of the incubation mixture containing 50 mM sodium acetate buffer, pH 4.5, 0.4 .mu.M FRET substrate, 2.4 nM enzyme, 5% DMSO, and 0.05% Brij-35. The tested compounds were serially diluted in DMSO and pre-incubated with the substrate. The reaction was started by addition of enzyme. |
| Affinity data for this assay | |
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