ReadiUse™ NADPH Regenerating Kit
NADPH provides the reducing equivalents for biosynthetic reactions and for oxidation-reduction involved in protection against the toxicity of ROS (reactive oxygen species). NADPH is also used for anabolic pathways, such as lipid synthesis, cholesterol synthesis and fatty acid chain elongation. It is the source of reducing equivalents for cytochrome 450 hydroxylation of aromatic compounds, steroids, alcohols, and drugs. NADPH is a necessary cofactor in many xenobiotic metabolism reactions. In chloroplasts, NADP is reduced by ferredoxin-NADP reductase in last step of the electron chain of the light reactions of photosynthesis. The NADPH produced is then used as reducing power for the biosynthetic reactions in the Calvin cycle of photosynthesis. Many oxidoreductases and all ligases use NADPH as coenzymes. NADPH is required for the measurement of oxidase activity catalyzed by P450s, FMOs, NADPH-P450 reductase, and many other oxidase enzymes. AAT Bioquest's RediUse™ NADPH Regenerating Kit provides two ready-to-use solutions to regenerate NADPH by a simple mixing. This kit can be used for all NADPH-requiring oxidase assays (cDNA-expressed enzymes and liver fractions). About 300-500 enzyme assays can be performed using this kit. The total number of assays that can be performed depends on a researcher's experimental design.
Example protocol
AT A GLANCE
Important notes
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
1. NADPH Regenerating stock solution (2X):
Make 2X NADPH Regenerating stock solution by adding the whole content of Assay Buffer II (Component B) and 500X Glucose-6-phosphate dehydrogenase (Component C) into Assay Buffer I (Component A). Mix well.
SAMPLE EXPERIMENTAL PROTOCOL
- Add equal volume of 2X NADPH Regenerating stock solution into the desired assay system. Note: 2.5 mL of Assay Buffer I (Component A), 2.5 mL of Assay Buffer II (Component B) and 10 µL of 500X Glucose-6-phosphate dehydrogenase (Component C) are enough for 1 plate.
Citations
View all 8 citations: Citation Explorer
Identification of osalmid metabolic profile and active metabolites with anti-tumor activity in human hepatocellular carcinoma cells
Authors: Wu, Zhe and Zhan, Yaqiong and Wang, Li and Tong, Jiepeng and Zhang, Li and Lin, Mengjia and Jin, Xuehang and Jiang, Lushun and Lou, Yan and Qiu, Yunqing
Journal: Biomedicine \& Pharmacotherapy (2020): 110556
Authors: Wu, Zhe and Zhan, Yaqiong and Wang, Li and Tong, Jiepeng and Zhang, Li and Lin, Mengjia and Jin, Xuehang and Jiang, Lushun and Lou, Yan and Qiu, Yunqing
Journal: Biomedicine \& Pharmacotherapy (2020): 110556
Functional characterization of CYP4G11–a highly conserved enzyme in the western honey bee Apis mellifera
Authors: Calla, Bernarda and MacLean, Marina and Liao, Ling-Hsiu and Dhanjal, Inderpreet and Tittiger, Claus and Blomquist, Gary J and Berenbaum, May R
Journal: Insect molecular biology (2018)
Authors: Calla, Bernarda and MacLean, Marina and Liao, Ling-Hsiu and Dhanjal, Inderpreet and Tittiger, Claus and Blomquist, Gary J and Berenbaum, May R
Journal: Insect molecular biology (2018)
Immunotoxic effects of benzo [a] pyrene on rainbow trout (Oncorhynchus mykiss)
Authors: Phalen, Laura Jane
Journal: (2014)
Authors: Phalen, Laura Jane
Journal: (2014)
exo-Brevicomin biosynthetic pathway enzymes from the Mountain Pine Beetle, Dendroctonus ponderosae
Authors: Song, Minmin and Delaplain, Patrick and Nguyen, Trang T and Liu, Xibei and Wickenberg, Leah and Jeffrey, Christopher and Blomquist, Gary J and Tittiger, Claus
Journal: Insect biochemistry and molecular biology (2014): 73--80
Authors: Song, Minmin and Delaplain, Patrick and Nguyen, Trang T and Liu, Xibei and Wickenberg, Leah and Jeffrey, Christopher and Blomquist, Gary J and Tittiger, Claus
Journal: Insect biochemistry and molecular biology (2014): 73--80
References
View all 14 references: Citation Explorer
High-level expression of recombinant glucose dehydrogenase and its application in NADPH regeneration
Authors: Xu Z, Jing K, Liu Y, Cen P.
Journal: J Ind Microbiol Biotechnol (2007): 83
Authors: Xu Z, Jing K, Liu Y, Cen P.
Journal: J Ind Microbiol Biotechnol (2007): 83
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Authors: Devadas M, Liu Z, Kaneda M, Arai K, Matsukawa T, Kato S.
Journal: Neurosci Res (2001): 359
Authors: Devadas M, Liu Z, Kaneda M, Arai K, Matsukawa T, Kato S.
Journal: Neurosci Res (2001): 359
Improved assay of hepatic microsomal cholesterol 7 alpha-hydroxylase activity by the use of hydroxypropyl-beta-cyclodextrin and an NADPH-regenerating system
Authors: Souidi M, Parquet M, Lutton C.
Journal: Clin Chim Acta (1998): 201
Authors: Souidi M, Parquet M, Lutton C.
Journal: Clin Chim Acta (1998): 201
Cellular antioxidant defense by a ubiquinol-regenerating system coupled with cytosolic NADPH-dependent ubiquinone reductase: protective effect against carbon tetrachloride-induced hepatotoxicity in the rat
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Journal: Biol Pharm Bull (1996): 1005
Authors: Takahashi T, Sugimoto N, Takahata K, Okamoto T, Kishi T.
Journal: Biol Pharm Bull (1996): 1005
Microsomal retinal synthesis: retinol vs. holo-CRBP as substrate and evaluation of NADP, NAD and NADPH as cofactors
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Authors: Napoli JL, Posch KC, Burns RD.
Journal: Biochim Biophys Acta (1992): 183
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