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Prepare Control:
purified BACE1 (1F) : Assay buffer (1D) 1:250
50 uL total: 0.2 uL 1F : 49.8 uL
Prepare substrates:
for BACE2 - component A2A:C 2C in 1:100 ratio
8x50uL = 400uL: 4uL 2A : 396 uL 2C
for BACE1 component A1A: D 1D in 1:100 ratio
12x50uL = 600uL: 6uL 1A : 594uL 1D
Prepare Reference standards:
DIlute reference standard 1:100 in assay buffer. Make subsequent 1:2 dilutions to make reference ladder. Do the same for BACE2 substrate?
put 50uL of tissue sample supernatant in each well. wait 10 minutes for everything to get to the same temperature.
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Measure 490/520nm every 5 minutes for 30-60 min. After add 50 uL of stop solution and measure again.