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ReadiUse™ NADPH Regenerating Kit

Chemical structure for ReadiUse™ NADPH Regenerating Kit
Chemical structure for ReadiUse™ NADPH Regenerating Kit
Ordering information
Price ()
Catalog Number15265
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


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.

Components


Component A: Assay Buffer I1 bottle (25 mL)
Component B: Assay Buffer II1 bottle (25 mL)
Component C: 500X Glucose-6-phosphate dehydrogenase (400 units/mL)1 vial (100 µL)

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

  1. 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
Specialized Enzymes in Insect Lipid Metabolism
Authors: MacLean, Marina Ann
Journal: (2019)
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)
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
exo-Brevicomin biosynthesis in the fat body of the mountain pine beetle, Dendroctonus ponderosae
Authors: Song, Minmin and Gorzalski, Andrew and Nguyen, Trang T and Liu, Xibei and Jeffrey, Christopher and Blomquist, Gary J and Tittiger, Claus
Journal: Journal of chemical ecology (2014): 181--189
Functional characterization of myrcene hydroxylases from two geographically distinct Ips pini populations
Authors: Song, Minmin and Kim, Amy C and Gorzalski, Andrew J and MacLean, Marina and Young, Sharon and Ginzel, Matthew D and Blomquist, Gary J and Tittiger, Claus
Journal: Insect biochemistry and molecular biology (2013): 336--343
An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis
Authors: Qiu, Yue and Tittiger, Claus and Wicker-Thomas, Claude and Le Goff, Gaelle and Young, Sharon and Wajnberg, Eric and Fricaux, Thierry and Taquet, Nathalie and Blomquist, Gary J and Feyereisen, René
Journal: Proceedings of the National Academy of Sciences (2012): 14858--14863

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
Changes in NADPH diaphorase expression in the fish visual system during optic nerve regeneration and retinal development
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
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
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
Authors: Napoli JL, Posch KC, Burns RD.
Journal: Biochim Biophys Acta (1992): 183
Absolute requirement for GTP in activation of human neutrophil NADPH oxidase in a cell-free system: role of ATP in regenerating GTP
Authors: Peveri P, Heyworth PG, Curnutte JT.
Journal: Proc Natl Acad Sci U S A (1992): 2494
The metabolism of prostaglandin D2. Evidence for the sequential conversion by NADPH and NAD+ dependent pathways
Authors: Robinson C, Herbert CA, Bedwell S, Shell DJ, Holgate ST.
Journal: Biochem Pharmacol (1989): 3267
Loss of NADPH during assays of HMG-CoA reductase: implications and approaches to minimize errors
Authors: Ness GC, Pendleton LC, Pendleton AS.
Journal: Lipids (1987): 409
Mechanism for the differential induction of mutation by S9 activated benzo[a]pyrene employing either a glucose-6-phosphate-dependent NADPH-regenerating system or an isocitrate-dependent system
Authors: Lindblad WJ, Jackim E.
Journal: Mutat Res (1982): 109
Quantitative requirements for NADPH in the support of aromatization by human placental microsomes
Authors: Sheean LA, Meigs RA.
Journal: Steroids (1981): 211