Description
NAD/NADH Assay Kit (Colorimetric) ab65348 provides a convenient and sensitive tool to quantify NAD+ and NADH, and measure their ratio, in samples from mammals and other species.
The NAD cycling enzyme mix in the kit specifically acts on NADH/NAD in an enzyme cycling reaction which significantly increases sensitivity and specificity. There is no requirement to purify NADH/NAD from samples.
The levels of both NADt (total NAD+ and NADH) and NADH can be easily measured; the level of NAD+ can be easily calculated by subtracting NADH from NADt. The assay is read by absorbance at 450 nm.
NAD / NADH assay protocol summary:
– extract samples from cells / tissues with extraction buffer and deproteinize with spin column
– for NADH measurement, heat samples to 60°C for 30 min to decompose NAD+, cool on ice (this step not necessary for measurement of total NAD+/NADH)
– add samples and standards to wells
– add reaction mix and incubate for 5 min at room temp to convert NAD to NADH
– add NADH developer and incubate for 1-4 hrs while reaction cycles
– analyze with microplate reader multiple times during the 1-4 hr incubation
– reaction can be stopped with stop solution.