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HTRF Biotin-APP-Eu cryptate (BACE1 substrate), 5000 Assay Points

APP substrate is labeled with both biotin and Eu3+ Cryptate and used to monitor human beta-secretase (BACE1) activity.

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Feature	Specification
Application	Protein Quantification

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APP substrate is labeled with both biotin and Eu3+ Cryptate and used to monitor human beta-secretase (BACE1) activity.

View product information

Product Variants

Part #:

64APBKAA

List Price

USD 1,863.10

Your online price:

Part #:

64APBKAB

List Price

USD 6,340.57

Your online price:

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).

Product information

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.

Specifications

Other Specifications

Application	Protein Quantification
Brand	HTRF
Detection Modality	HTRF
Product Group	Fluorescent Reagent
Shipping Conditions	Shipped in Dry Ice
Target Class	Biomarkers
Target Species	Human
Technology	TR-FRET
Unit Size	5,000 assay points

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How it works

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.

Assay validation

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