The traditional NAD/NADH and NADP/NADPH assays are done by monitoring the changes in NADH or NADPH absorption at 340 nm. These methods suffer low sensitivity and high interference since the assays are done in the UV range that requires expensive quartz microplates. The low sensitivity of the absorption-based NADP/NADPH assays makes the assays difficult to be automated for high throughput screening that often uses small sample size.


Key Features

Broad Application : Can be used for quantifying NADP/NADPH in solutions and in cell extracts.
Sensitive             : Detect as low as 10 picomoles of NADP/NADPH in solution.
Continuous         : Easily adapted to automation without a separation step.
Convenient          : Formulated to have minimal hands-on time.
Non-Radioactive  : No special requirements for waste treatment.

Kit Components

Component A : NADP/NADPH Recycling Enzyme Mixture        2 bottles (lyophilized powder)
Component B : NADPH Sensor Buffer                                    1 bottle (20 mL)
Component C : NADPH Standard                                           1 vial (167 μg)
Component D : NADPH Extraction Solution                             1 bottle (10 mL)
Component E : NADP Extraction Solution                               1 bottle (10 mL)
Component F : NADP/NADPH Control Solution                        1 bottle (10 mL)
Component G : NADP/NADPH Lysis Buffer                             1 bottle (10 mL)



Brief Summary

→ Prepare 25 μL of NADPH standards and/or test samples
→ Add 25 μL of NADPH or NADP Extraction Solution
→ Incubate at room temperature for 15 minutes
→ Add 25 μL of NADP or NADPH Extraction Solution (25 μL)
→ Add 75 μL of NADP/NADPH reaction mixture
→ Incubate at RT for 15 minutes to 2 hours
→ Monitor fluorescence intensity at Ex/Em = 540/590 nm

References

1. Ziegenhorn J, Senn M, Bucher T. (1976) Molar absorptivities of beta-NADH and beta-NADPH. Clin Chem, 22, 151.
2. Ikegami T, Kameyama E, Yamamoto SY, Minami Y, Yubisui T. (2007) Structure and Properties of the Recombinant
    NADH-Cytochrome b(5) Reductase of Physarum polycephalum. Biosci Biotechnol Biochem.
3. Kimura N, Fukuwatari T, Sasaki R, Shibata K. (2006) Comparison of metabolic fates of nicotinamide, NAD+ and
    NADH administered orally and  intraperitoneally; characterization of oral NADH. J Nutr Sci Vitaminol (Tokyo), 52, 142.
4. O'Donnell JM, Kudej RK, LaNoue KF, Vatner SF, Lewandowski ED. (2004) Limited transfer of cytosolic NADH into
    mitochondria at high cardiac workload. Am J Physiol Heart Circ Physiol, 286, H2237.