Assay Method Information

Assay Name:  Activity Assay
Description:  A BACE2 activity assay kit from AnaSpec (Fremont, Calif.) was used to determine the potency of the selected compounds by AnaSpec BACE2 fluorescent assay. To each well of a black 96-well micro plate, 50 μl of the β-secretase substrate (Hylite Fluor 488) diluted 1:100 using assay buffer was first added.The compounds were serially diluted (1:2) into DMSO from their 10 mM stock concentrations in DMSO. The serially diluted inhibitors were diluted 1:100 into 1× assay buffer in a 96-well polypropylene plate (assay dilution plate). The final concentration of these compounds tested was 300 nM, 150 nM, 75 nM, 37.5 nM, 18.75 nM, 9.375 nM, 4.688 nM, 2.344 nM, 1.172 nM, 0.586 nM, and 0.293 nM.A volume of 10 μl of the inhibitor from the assay dilution plate, a control inhibitor LY2886721 or inhibitor vehicle was then added to each well of the black 96-well micro plate.The recombinant BACE2 was diluted in an assay buffer and the final BACE2 was 10.6 nM. The enzyme was added in a volume of 40 μl to each well of a black 96-well micro plate.The reagents were mixed gently and the assay plate was placed into a POLARstar fluorescent plate reader with excitation wavelength set at 485 nm and the emission wavelength set at 520 nm. The plate reader was set to take readings every 5 minutes for up to 30 minutes or a 30 minute endpoint reading was used.
Affinity data for this assay
 

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