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I had another question about the Caspase 9 Inhibitor Drug Detection Kit (ab102497). I have run the assay and the RFUs for the positive inhibition are about 10x the RFUs for the background control. Is this expected? I have not tried higher concentrations of VAD-FMK; should the concentration of VAD-FMK specified in the protocol fully inhibit caspase 9 activity? Which control would you suggest using for normalization?
Asked on Oct 02 2012
Thank you very much for your email and for your patience while I've been in touch with the lab regarding your enquiry.
My contact at the lab has informed me that the background control is the fluorescence emitted by the substrate and the reaction buffer. These values have to be subtracted from all values. Your signals from the active caspase activity should be taken as 100% activity and used for normalization. The signals you then get from the use of our caspase inhibitor will give you an idea of how inhibition looks and what kind of signals it produces. This can be reported in terms of % inhibition. Then you can use your experimental inhibitor to looks at the % inhibition it produces.
I hope that this information will be useful, but if you have further questions or need anything else, please let me know and I'll be happy to help.
Answered on Oct 02 2012