I have been using the universal SIRT activity assay from Abcam (http://www.abcam.com/universal-sirt-activity-assay-kit-colorimetric-ab156915.html) to measure SIRT activity in nuclear extracts from mouse brain. I seem to be having problems with my OD duplicates, that they are quite far away from one another. For example:
0.829 0.632
0.853 0.688
The no SIRT co-factor control (i.e. no NAD+) and the blanks, however, do not appear to have this issue (see duplicates below).
0.092 0.083
0.100 0.118
I have been incredibly meticulous when pipetting, and have also been sure to add reagents sequentially and in the same order each time e.g. adding developer and stop solution to wells in the same order. I wondered if it might have something to do with the nuclear extracts I am using? They are, after all, in a lysis buffer - I'm not sure if this might affect the assay? Do any of you have experience using this kit with nuclear extracts? If so, I would love to hear from you.
Thank you in advance!
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