AnaSpec, EGT Group, the provider of ultra-sensitive
fluorimetric SensoLyte® line of assay kits is pleased to announce the release
of the SensoLyte® Fluorimetric NADP/NADPH Assay Kit. This assay is
a sensitive fluorimetric assay that detects NADP and NADPH without interfering
with NAD/NADH. NADP is converted to NADPH in an enzyme-cycling reaction. The
kit contains a fluorogenic reagent, producing fluorescence when reacted with
NADPH. The resulting red fluorescence can be monitored at excitation /emission=
560/590 nm, with the intensity of fluorescence produced being proportional to
NADP/NADPH concentration. This assay detects as low as 1.5 nM of the analyte.
Pyridine nucleotides are involved in a number
of critical catabolic and anabolic reactions in living organisms. Nicotinamide
adenine dinucleotide phosphate (NADP) is a natural coenzyme present in all animal
and plant organisms. Cellular NADPH is important for tolerance to ROS and maintenance
of cellular redox homeostasis.1-5 The reducing power of NADPH is
a required cofactor for enzymes that are prooxidant such as nitric oxide synthase
and NADPH oxidase.6 Furthermore the NADP+/NADPH ratio has been found
to change in the erythrocytes of subjects affected by hemolytic disorder.7,
8
In addition to the fluorimetric kit, AnaSpec also
provides a convenient colorimetric kit. This kit also detects NADP and NADPH
without interfering with NAD/NADH. The SensoLyte® Colorimetric NADP/NADPH
Assay Kit utilizes an enzyme cycling reaction to produce a blue colored product,
formazan, which can be monitored at 565 nm using a microplate reader. The intensity
of the color produced is proportional to NADP/NADPH concentration.
Fig 1. NADPH and NADH were serially diluted in assay buffer containing Enzyme
Cycling Mix and Detection Reagent, and fluorescence was recorded at Ex/Em=570
/590 nm. (Flexstation 384II, Molecular Devices).
Fig 2. NADP and NAD were serially diluted in assay buffer containing Enzyme
Cycling Mix and Detection Reagent, and fluorescence was recorded at Ex/Em=570/590
nm. (Flexstation 384II, Molecular Devices).
References:
1. Holmgren, A. J.
Biol. Chem. 264, 13963 (1989).
2. Grant, CM. et al. Mol. Microbiol 21, 171 (1996a).
3. Muller, EG. et al. Mol.
Biol.Cell 7, 1805 (1996).
4. Ng, CH. et al. Free
Radic. Biol. Med. 44, 1131 (2008).
5. Temple, MD. et al. Trends Cell Biol. 15, 319 (2005).
6. Stamler, JS. et al. Science
258, 1898 (1992).
7. Kirkman, HN. et al.
J. Clin. Invest. 55, 875 (1975).
8. Magnani, M. Acta Haematol.
75, 211 (1986).
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