I obtained the EnzChek peptidase/protease assay kit. I carried out the assay according to the manufacturer's instruction using a microplate and read at excitation/emmission of 490/520 but i got OVERFLW readings even for the negative control. Anyone with prior experience?
What was in your negative control well? Any of the fluorescent substrate? What kind of plate are you using (wells, plastic/glass/opaque bottom, clear/opaque wells, etc.)?
I used a RediPlate EnzChek red (BODIPY TR-X dye) kit and a TECAN reader to test some bacterial proteases. It took 10 to 20 runs to find the right conditions for this particular combination of clear flat bottom tube and battered reader. To find the right filter set and define all appropriate parameters on the reader I diluted the substrate in eight steps from 0 to 2.5µM (according to the manual) and added Proteinase K to digest the casein. By adjusting the number of flashes, flash duration and gain value I was able to quickly find the right conditions. Good luck!
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