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Total Solution for NAD/NADH & NADP/NADPH Detection


Total NAD and NADH dose responses were measured with Amplite® Colorimetric Total NAD and NADH Assay Kit in a 96-well white/clear bottom plate. As low as 0.1 µM total NAD and NADH was detected with 1 hour incubation (n=3). A: Absorbance measured at 460 nm, B: Absorbance measured at 635 nm after adding enhancer to each well.
Nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) are two important cofactors found in cells. NADH is the reduced form of NAD+ and NAD+ is the oxidized form of NADH. NAD forms NADP with the addition of a phosphate group to the 2' position of the adenyl nucleotide through an ester linkage. NADP is used in anabolic biological reactions, such as fatty acid and nucleic acid synthesis, which require NADPH as a reducing agent. In chloroplasts, NADP is an oxidizing agent important in the preliminary reactions of photosynthesis. The NADPH produced by photosynthesis is then used as reducing power for the biosynthetic reactions in the Calvin cycle of photosynthesis. The traditional NAD/NADH and NADP/NADPH assays are based on monitoring absorption changes in NADH or NADPH at 340 nm. The short UV wavelengths of NAD/NADH and NADP/NADPH assays make the traditional methods suffer low sensitivity and high interference.

AAT Bioquest offers the most comprehensive product portfolio for NAD/NADH and NADP/NADPH detection as summarized in Table 1. All our NAD/NADH and NADP/NADPH assay kits are in a mix and read format with minimal hands-on time required. These kits have either significantly improved sensitivity and dynamic range or less interference from biological samples compared to the commercial assay kits from other vendors. Through our continuous improvement and innovation, our new NAD/NADH and NADP/NADPH assay kit 15275 and kit 15276 achieve outstanding sensitivity.

Amplite® Colorimetric Total NAD and NADH Assay Kit provides a convenient method for detecting total NAD and NADH. There is no need to purify NAD and NADH from sample mix. The enzymes in the system specifically recognize NAD and NADH in an enzyme cycling reaction, and thus significantly increase detection sensitivity. The NAD/NADH probe is a chromogenic sensor that has its maximum absorbance at 460 nm upon NAD/NADH reduction. The maximum absorbance increases to ~635 nm when enhancer is added to the assay system. The absorption of the NAD/NADH probe is directly proportional to the concentration of NAD/NADH. Amplite® Colorimetric Total NAD and NADH Assay Kit detects as little as 0.1 µM total NAD and NADH in a 100 µL assay volume. Compared to Amplite® Colorimetric Total NAD and NADH Assay Kit 15258, Kit 15275 demonstrates higher sensitivity.
 

Table 1. NAD/NADH & NADP/NADPH Assay Comparison

Cat#
Product Name
Ex (nm)
Em (nm)
Detection Limit
Dynamic Range
Unit Size
15259Amplite® Fluorimetric Total NADP and NADPH Assay Kit *Red Fluorescence*5405900.01 µM 0-3 µM 400 Tests
15260Amplite® Colorimetric Total NADP and NADPH Assay Kit575 0.1 µM 0-3 µM 400 Tests
15262Amplite® Fluorimetric NADPH Assay Kit *Red Fluorescence*5715851 µM 0-100 µM 400 Tests
15264Amplite® Fluorimetric NADP/NADPH Ratio Assay Kit *Red Fluorescence*5715850.01 µM 0-3 µM 250 Tests
15272Amplite® Colorimetric NADPH Assay Kit460 3 µM 1-200 µM 400 Tests
15274Amplite® Colorimetric NADP/NADPH Ratio Assay Kit460 0.03 µM 0.03-1 µM 250 Tests
15276Amplite® Colorimetric Total NADP and NADPH Assay Kit *Enhanced Sensitivity*460 0.03 µM 0.03-1 µM 400 Tests
15281Amplite® Fluorimetric NADP Assay Kit *Blue Fluorescence*4224660.03 µM 0.03-1 µM 200 Tests
15257Amplite® Fluorimetric Total NAD and NADH Assay Kit *Red Fluorescence*5715850.1 µM 0-3 µM 400 Tests
15258Amplite® Colorimetric Total NAD and NADH Assay Kit575 0.3 µM 0-10 µM 400 Tests