W tried to determine the NADtotal and NADH on skeletal muscle of mice (gastrocnemius). Unfortunately, we obtain the same values for NADtotal and NADH that suggest we do not have NAD in our sample that is not possible. We don't have any idea about what we've done wrong.
For the homogenization, we crushed muscle into powder and we add 400uL of extraction buffer. We don't wash the muscle into PBS because the muscle were frozen. Could be that the issue ? Should we deproteinized before use in assay by filtering through a 10 kDa cut-off spin filter ?
Thanks
For metabolite assays (e.g., for ATP, CP, NADH), most would typically deproteinize muscle samples with perchloric acid at near freezing temperatures. I have never seen a cut-off spin filter used for deproteinization but then again, I don't deproteinize muscle samples that often.