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[cmel]

Hi,

I'm trying to set up a protease assay which can be used to screen for inhibitors of BACE (beta secretase). The paper i'm using is Anal Bioanal Chem 2007 388:1175-1183.

I can't see anything that i'm doing different to them, but my assay is just not working! help!

My protocol:

Assay buffer is 50 mM Na Acetate, pH 4.5
Stop Buffer is 2.5 M Na Acetate, pH 8

I add casein-FITC (20 uL of varying concentrations - at the moment ranging from 1-100 ug/mL)
then 20 ul assay buffer,
and 20 ul BACE (total of 10 mU/well - in the paper they use 6.6 mU/well)

The plate is incubated for 1 hour, 37oC, Then I add 20 ul stop buffer and let it sit for 5 minutes before reading at 485/538 nm

I do the assay in a white 96 well plate (I don't have any black plates handy)

Please help, it's driving me crazy!

I've even made fresh buffers, double and triple checked them. I really don't know what i'm doing wrong.

The BACE is a fresh batch, and so is the Casein-FITC.