Amplite® Colorimetric Total NADP and NADPH Assay Kit Enhanced Sensitivity

Name: Amplite® Colorimetric Total NADP and NADPH Assay Kit *Enhanced Sensitivity*
Brand: AAT Bioquest
SKU: 15276
Price: 341 USD
Availability: InStock

Nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) are two important cofactors found in cells. NADH is the reduced form of NAD+, and NAD+ is the oxidized form of NADH. It forms NADP with the addition of a phosphate group to the 2' position of the adenyl nucleotide through an ester linkage. NADP is used in anabolic biological reactions, such as fatty acid and nucleic acid synthesis, which require NADPH as a reducing agent. In chloroplasts, NADP is an oxidizing agent important in the preliminary reactions of photosynthesis. The NADPH produced by photosynthesis is then used as reducing power for the biosynthetic reactions in the Calvin cycle of photosynthesis. The traditional NAD/NADH and NADP/NADPH assays are done by monitoring of NADH or NADPH absorption at 340 nm. This method suffers low sensitivity and high interference since the assay is done in the UV range that requires expensive quartz microplate. This Amplite® NADP/NADPH Assay Kit provides a convenient method for sensitive detection of NADP and NADPH. The enzymes in the system specifically recognize NADP/NADPH in an enzyme cycling reaction. There is no need to purify NADP/NADPH from sample mix. The enzyme cycling reaction significantly increases detection sensitivity. Compared to Kit #15260, this kit has higher sensitivity.

Example protocol

AT A GLANCE

Protocol Summary

Prepare NADPH standards or test samples (50 µL)
Add NADP/NADPH working solution (50 µL)
Incubate at room temperature for 15 minutes to 2 hours
Monitor Absorbance at 460 nm

Important Thaw one of each kit component at room temperature before starting the experiment.

CELL PREPARATION

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

PREPARATION OF STOCK SOLUTIONS

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.

NADPH standard solution (1 mM)

Add 200 µL of 1X PBS buffer into the vial of NADPH Standard (Component C) to make 1 mM (1 nmol/µL) NADPH standard solution.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/15276

NADPH standard

Add 2 µL of 1 mM NADPH standard solution into 998 µL 1X PBS buffer (pH 7.4) to generate 2 µM (2 pmols/µL) NADPH standard solution (NS7). Take 2 µM NADPH standard solution (NS7) and perform 1:2 serial dilutions in 1X PBS buffer to get serially diluted NADPH standards (NS6 - NS1). Note: Diluted NADPH standard solution is unstable, and should be used within 4 hours.

PREPARATION OF WORKING SOLUTION

Add 8 mL of NADPH Probe Buffer (Component B-II) to the bottle of NADP/NADPH Recycling Enzyme Mix (Component A) and mix well.
Add 2 mL of NADPH Probe (Component B-I) into the bottle of Component A+B-II and mix well to make NADP/NADPH working solution.
Note This NADP/NADPH working solution is enough for 200 assays.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of NADPH standards and test samples in a white/clear bottom 96-well microplate. NS= NADPH Standards (NS1 - NS7, 0.0313 to 2 µM), BL=Blank Control, TS=Test Samples.

BL	BL	TS	TS
NS1	NS1	...	...
NS2	NS2	...	...
NS3	NS3
NS4	NS4
NS5	NS5
NS6	NS6
NS7	NS7

Table 2. Reagent composition for each well. High concentration of NADPH (e.g., >30 µM, final concentration) will cause a saturated signal and make the calibration curve non-linear.

Well	Volume	Reagent
NS1 - NS7	50 µL	Serial Dilutions (0.0313 to 2 µM)
BL	50 µL	1X PBS buffer
TS	50 µL	test sample

Prepare NADPH standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
Note Prepare cells or tissue samples as desired. Lysis Buffer (Component D) can be used for lysing the cells for convenience.
Add 50 µL of NADP/NADPH working solution to each well of NADPH standard, blank control, and test samples to make the total NADP/NADPH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of NADP/NADPH working solution into each well instead, for a total volume of 50 µL/well.
Incubate the reaction at room temperature for 15 minutes to 2 hours, protected from light.
Monitor the absorbance increase with an absorbance plate reader at 460 nm.

Citations

View all 60 citations: Citation Explorer

Synergistic and stepwise treatment of resveratrol and catechol in Haematococcus pluvialis for the overproduction of biomass and astaxanthin
Authors: Qiu, Jia-Fan and Yang, Yu-Cheng and Li, Ruo-Yu and Jiao, Yu-Hu and Mou, Jin-Hua and Yang, Wei-Dong and Lin, Carol Sze Ki and Li, Hong-Ye and Wang, Xiang
Journal: Biotechnology for Biofuels and Bioproducts (2024): 80

The Linkage of Yeast Metabolites, Produced Under Hyperosmotic Stress, to Cellular Cofactor Systems During Icewine Fermentation.
Authors: Allie, Robert
Journal: (2022)

Enhanced 1, 3-propanediol production in Klebsiella pneumoniae by a combined strategy of strengthening the TCA cycle and weakening the glucose effect
Authors: Lu, Xinyao and Ren, Shunli and Lu, Jingzheng and Zong, Hong and Song, Jian and Zhuge, Bin
Journal: Journal of applied microbiology (2018)

Cytosolic Redox Status of Wine Yeast (Saccharomyces Cerevisiae) under Hyperosmotic Stress during Icewine Fermentation
Authors: Yang, Fei and Heit, Caitlin and Inglis, Debra L
Journal: Fermentation (2017): 61

Pyrroloquinoline Quinone, a Redox-active o-Quinone, Stimulates Mitochondrial Biogenesis by Activating SIRT1/PGC-1α Signaling Pathway
Authors: Saihara, Kazuhiro and Kamikubo, Ryosuke and Ikemoto, Kazuto and Uchida, Koji and Akagawa, Mitsugu
Journal: Biochemistry (2017)

Page updated on November 23, 2024

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