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Assay Method Information

Assay Name:  Fluorescent-Peptide Cleavage Assay
Description:  BACE2 enzyme ectodomain (derived from plasmid pET17b-T7-hu proBACE2) was prepared as described in Ostermann et al., Crystal Structure of Human BACE2 in Complex with a Hydroxyethylamine Transition-state Inhibitor, Journal of Molecular Biology 2006, 355, 249-261. The pro-enzyme was stored at 4 C. at a concentration of 70 ug/ml.A fluorescent peptide with the amino acid sequence WS EVNLD AEFRC-MR121 was synthesised and a stock solution of 1.5 mM in DMSO prepared and stored at -20 C. MR121 is a fluorophore with an excitation wavelength of 630 nm and emission wavelength of 695 nm. The MR121 fluorescence is quenched by the N-terminal tryptophan until the peptide is cleaved by BACE2.Assays were all made in a Corning 384-well black polystyrene non-binding surface microtitre plate with clear flat bottom and using a Plate:Vision (Perkin Elmer) fluorescence reader. To perform the assay an 80 nM solution of BACE2 was prepared in assay buffer.
Affinity data for this assay
 

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Last update November 1, 2007
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