Development of the HTRF assay
Biotin-APP-Eu cryptate (BACE1 substrate) HTRF®
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No-wash
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Ease-of-use
Overview
Beta-secretase (BACE1) is an enzyme which cleaves amyloid precursor protein (APP) to produce the amyloid ß42 peptide, responsible for insoluble amyloid plaques in Alzheimer's disease (AD).
HTRF APP substrate labeled with both biotin and Eu3+ Cryptate, used with HTRF SA-XL665 or Sa-d2, provides a sensitive and efficient method for screening BACE1 inhibitors with the robustness of HTRF technology.
Benefits
- BACE1 ACTIVITY
- BATCH-TO-BATCH REPRODUCIBILITY
- ALLOWS HOMOGENEOUS ASSAY
Assay principle
Assay protocol
Evaluation of BACE1 peptidic inhibitors
The HTRF BACE1 assay was used to compare the activity of soluble proBACE1 (sproBACE1) and a soluble BACE1 that was autoprocessed to remove the prodomain sequence (sautoBACe1), in the presence of selected inhibitors. 25 nM Biotin-APP substrate-Eu cryptate, 5 µM unlabeled APP peptide substrates and 6 nM enzyme were incubated for 3 h at 30 °C, pH 5.0, in the presence and absence of increasing concentrations of peptidic inhibitors.
IC50s included in Table below and are in good agreement with those reported in the literature using kinetic assays (Kennedy ME et al, Anal Biochem, 2003 Aug 1;319(1):49-55.). sproBACE1 (¦) and sautoBACE1 (°) KTEEISEVNL-(statine)-VAEF (- - - - -) EVNL(GHET)AAEF (—) ELDL(GHET)AVEF (———)
Peptide inhibitor | IC50 for sproBACE1 (nM) | IC50 for sautoBACE1 (nM) |
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KTEEISEVN-(statine)-VAEF | 92 ± 9 | 82 ± 18 |
EVNL(?HET)AAEF | 8 ± 3 | 6 ± 2 |
ELDL(?HET)AVEF | 4 ± 1 | 4 ± 1 |
IsoValeryl-VV-statine-A-statine | n.d | n.d |
HTRF PPI your dream assay served on a plate
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All your HTRF assays in one document! - Catalog
A guide to Homogeneous Time Resolved Fluorescence
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Get the brochure about technology comparison. - Brochures
Virology research solutions using HTRF Protein-Protein Interaction assays
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Plate Reader Requirement
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