I am using the Promega ADP Glo assay to look at inhibitors. I am getting a very large amount of noise in the assay. The noise is so bad that I cant reliably measure compound IC50s.
I wonder if anyone has had this issue with the Kit and perhaps you can share any solutions you found?
Paul
I have seen this problem, too. I think the reason is that the ATP in the enzyme reaction was not completely destroyed in the first step of the detection in some wells. Tiny amounts may have been left clinging to the walls of the wells. When the second step reagent is added to convert ADP to ATP, the volume increases and this residual ATP is also detected. Centrifugation of the plates after each addition did not seem to help very much, but it is worth a try if you have a plate centrifuge.
A possible solution would be to use shallow-well, low-volume 384-well plates rather than deep-well, low-volume 384-well plates, and to make sure that no liquid is left on the sides of the wells after addition of the first detection reagent. However, this is tricky because of the relatively large volumes of detection reagents that have to be added.
Another detection kit you could use is Bellbrook's Transcreener.
http://www.bellbrooklabs.com/PDFs/Tech%20Man_ADP2%20FP%20EZ_v073009.pdf
This is a fluorescence polarization assay. I used the AMP detection kit and it worked quite well. It is also expensive, so it is forces you to minimize the reaction volumes.
I haven't used that specific plate, but it is a shallow-well plate of the type I suggested. Good luck.
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