BACE1 Assay Kit

Use a rapid, sensitive and cost effective alternative to ELISA

HTRF BACE1  assay provides a sensitive and efficient method for screening BACE1  (or human b-secretase) inhibitors in a high-throughput manner. This assay uses a new HTRF APP swedish peptide substrate.

 

BACE1 cleaves amyloid precursor protein (APP) to produce the amyloid β42 peptide which is responsible for  insoluble amyloid plaques of Alzheimer's disease (AD). The development of specific BACE-1 inhibitors has therefore become an attractive approach for treatment of AD.

 

Assay principle

BACE1 assay uses an 18-aminoacid APP Swedish peptide substrate labeled with europium cryptate on one end and biotin on the other (biotin-APP substrate-K). The FRET signal is generated with the addition of SA-XL665.  In the presence of BACE1, the APP substrate is cleaved, causing a decrease in signal as FRET is lost.

The  assay is run in only two steps.  The reaction is initiated by the addition of APP substrates (unlabeled and cryptate labeled) and is terminated with the addition of streptavidin labeled with XL665 or d2 diluted in detection buffer. To reach maximum signal, the concentration of each assay component needs  to be optimized. The figure to the right describes the assay procedure in 20µL final.

Assay performance

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.

Fig 1: The inhibition curves for KTEEISEVNL-(statine)-VAEF (- - - - -), EVNL(ΓHET)AAEF (—), and ELDL(ΓHET)AVEF (———) at sproBACE1 (■) and sautoBACE1 (°). Reactions were run in the presence of 25 nM EuK-APPsw and 5 µM unlabeled APPsw peptide substrates and 6 nM enzyme in the presence and absence of increasing concentrations of peptidic inhibitors for 3 h at 30 °C, pH 5.0. The ratio values measured at each concentration were normalized to the maximum ratio (no enzyme) and minimum ratio (3 h time point) to give percentage inhibition values. IC50s are included in Table 1 and are in good agreement with those reported in the literature using kinetic assays (Kennedy ME et al, Anal Biochem, 2003)

Peptide inhibitor IC50 for sproBACE1 (nM) IC50 for sautoBACE1 (nM)
KTEEISEVN-(statine)-VAEF 92 ± 9 82 ± 18
EVNL(ψHET)AAEF 8 ± 3 6 ± 2
ELDL(ψHET)AVEF 4 ± 1 4 ± 1
IsoValeryl-VV-statine-A-statine n.d n.d

IC50 values represent the mean ± SD from three to four determinations; n.d., no inhibition detected

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Biotin-APP substrate-Eu cryptate - 5,000 tests64APBKAA
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Biotin-APP substrate-Eu cryptate- 20,000 tests64APBKAB
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APP substrate - 5,000 tests64APPKAA
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APP substrate - 20,000 tests64APPKAB
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