Hi. I am recently trying to measure NAD+ from E2ND-100 kit from bioassay but I can't get any consistent results.
This is my protocol.
1. Prepare 2 x 105 of A549 cell pellet washed with DPBS 2 times. (I have control group, vehicle group and sample group that has been treated with the drug believed to increase cellular NAD+ level)
2. Add 100ul NAD extraction buffer from kit and lysis the cell by freezing and thawing method.(Snap freeze in Liquid nitrogen and thaw on ice for 10min x 2 cycles)
3. Incubate at 60℃ for 15min.
4. Add 20ul assay buffer and 100ul NADH extraction buffer.
5. Briefly votex and centrifuge for 14,000rpm, 4℃, 5min.
6. Normalized the sample with BCA assay. Add mixture of NAD extraction buffer, NADH extraction buffer and assay buffer with ratio of 5:5:1 to make total 90ul.
7. Put 40ul of samples and standards into 96well.
8. Add reaction buffer and read the plate at 565nm.
9. After incubation on R.T, read the plate at 565nm.
Although the kit protocol says Incubate at 60℃ for 5 min, I increased the time in case 5 min might short to degrade NADH.
Please help me with my assay. Thank you.
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