Assay Method Information | |
| Fluorescence Quenching Assay (FRET) |
Description: | For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACE.sub.ect. cells into OptiMEM.TM. (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/ml in DMSO.The assay was performed in the presence of 5 .mu.l OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 .mu.l water containing the desired concentration of test compound and 1% DMSO, 1 .mu.M substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 .mu.l in a 384 well plate. In general, 25 .mu.l of compound dilution were given to the plate followed by the addition of 10 .mu.l of BACE. |
Affinity data for this assay | |
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