HTRF Human APP (BACE1 Substrate) Biotin Eu-Conjugate, 5,000 Assay Points

Assay principle

The HTRF BACE1 assay is based on our amyloid precursor protein (APP) substrate, labeled with both biotin and Eu3+ Cryptate. Active BACE1 enzyme cleaves the substrate, causing the loss of energy transfer, and thus a reduction in SA-XL665 or Sa-d2 emissions.

Assay protocol

The assay is run in a two-step process: 1.BACE1 enzymatic reaction: Mix together the APP substrate, BACE1 and/or compounds, then incubate (we recommend a time course study to determine the optimal stimulation time). 2.BACE1 activity detection: Add Streptavidin-XL665 or Streptavidin-d2.

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