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NADP is the electron acceptor for biosynthetic reactions and dehydrogenation reactions. NADP is used for anabolic pathways, such as lipid synthesis, cholesterol synthesis and fatty acid chain elongation. It is also a necessary cofactor in many xenobiotic metabolism reactions. In chloroplasts, NADP is reduced to NADPH by ferredoxin-NADP reductase in the last step of the electron chain in photosynthesis reactions. Many oxidoreductases and all ligases use NADP/NADPH as coenzyme. NADP can also be used in the determination of amylase, creatine kinase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase. AAT Bioquest's ReadiUse™ NADP Regenerating Kit provides a flexible prepackaged NADP regenerating system to accommodate various experimental designs. It uses two ready-to-use solutions to regenerate NADP by a simple mixing. This kit can be used for all NADP-requiring systems (such as: 12-Ketochenodeoxycholic Acid synthesis, Ferredoxin Reductase System). 300-500 enzyme assays can be performed using this kit.

Example protocol

AT A GLANCE

Important notes
Thaw all the components at room temperature before use.

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 solution (40X):
Add 0.5 mL H₂O into one vial of NADPH (Component D) to make 40X NADPH solution. Note: One vial of NADPH is for making 10 mL NADP Regenerating Solution.

2. NADP Regenerating Enzyme Mix solution (400X):
Add 100 µL H₂O into the vial of NADP Regenerating Enzyme Mix (Component C) to make 400X NADP Regenerating Enzyme Mix solution. Protect from light.

PREPARATION OF WORKING SOLUTION

Add 5 mL of NADP Regenerating Buffer I (Component A), 5 mL of NADP Regenerating Buffer II (Component B), 0.5 mL of 40X NADPH solution, and 50 μL of 400X NADP Regenerating Enzyme Mix solution to make a total volume of 10.55 mL 2X NADP regenerating solution. Keep from light. Note: Activated NADP regenerating solution is not stable, and should be prepared freshly before use. 10 mL of NADP Regenerating Solution is enough for 1 plate.

SAMPLE EXPERIMENTAL PROTOCOL

Add equal volume of 2X NADP Regenerating solution into the desired assay system.

Citations

View all 4 citations: Citation Explorer

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

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

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

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