| Assay Method Information | |
| | Homogeneous Time-Resolved FRET Assay |
| Description: | The protocol that was used to determine the recited values isdescribed as follows.BACE1 HTRF FRET AssayReagentsNa+-Acetate pH 5.01% Brij-35GlycerolDimethyl Sulfoxide (DMSO)Recombinant human soluble BACE1 catalytic domain (>95% pure)APP Swedish mutant peptide substrate (QSY7-APPswe-Eu):QSY7-EISEVNLDAEFC-Europium-amideA homogeneous time-resolved FRET assay was used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitored the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contained an N-terminal QSY7 moiety that served as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence was low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme. |
| Affinity data for this assay | |
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