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Amplite® Fluorimetric NADPH Assay Kit *Red Fluorescence*

NADPH dose response was measured with Amplite® Fluorimetric NADPH Assay Kit in a 96-well solid black plate using a NOVOStar microplate reader (BMG Labtech). RFU measured over Ex/Em = 540/590 nm.
NADPH dose response was measured with Amplite® Fluorimetric NADPH Assay Kit in a 96-well solid black plate using a NOVOStar microplate reader (BMG Labtech). RFU measured over Ex/Em = 540/590 nm.
Ordering information
Price ()
Catalog Number15262
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

OverviewpdfSDSpdfProtocol


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® NADPH Assay Kit provides a convenient method for sensitive detection of NADPH. The enzymes in the system specifically recognize NADPH in an enzyme cycling reaction. The enzyme cycling reaction significantly increases detection sensitivity.

Platform


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Recommended plateSolid black

Components


Component A: NADPH Recycling Enzyme Mixture2 bottles (lyophilized powder)
Component B: NADPH Assay Buffer1 bottle (20 mL)
Component C: NADPH Standard1 vial (167 µg)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare NADPH working solution (50 µL)
  2. Add NADPH standards or test samples (50 µL)
  3. Incubate at room temperature for 15 minutes - 2 hours
  4. Monitor fluorescence increase at Ex/Em = 540/590 nm

Important notes
Thaw one of each kit component 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 standard solution (1 mM):
Add 200 µL of PBS buffer into the vial of NADPH standard (Component C) to make 1 mM (1 nmol/µL) NADPH stock solution.

PREPARATION OF STANDARD SOLUTION

NADPH standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15262

Use NADPH standard solution and PBS buffer (pH 7.4) to generate 100 µM (100 pmol/µL) NADPH standard solution (NS7). Then use 100 µM NADPH standard solution to perform 1:3 serial dilutions to get remaining serial dilutions of NADPH standard (NS6 - NS1). Note: Diluted NADPH standard solution is unstable, and should be used within 4 hours.

PREPARATION OF WORKING SOLUTION

Add 10 mL of Amplite™ NADPH Assay Buffer (Component B) into the bottle of NADPH Recycling Enzyme Mixture (Component A); mix well. Note: This NADPH working solution is enough for two 96-well plates. The working solution is not stable, use it promptly and avoid direct exposure to light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of NADPH standards and test samples in a solid black 96-well microplate. NS = NADPH standard (NS1 - NS7, 0.1 to 100 µM); BL = blank control; TS = test sample.

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

Well Volume Reagent
NS1 - NS7 50 µL Serial Dilution (0.1 to 100 µM)
BL 50 µL PBS
TS 50 µL Test Sample
  1. Prepare NADPH standards (NS), blank controls (BL), and test samples (TS) into a solid black 96-well microplate according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Prepare cells or tissue samples as desired.

  2. Add 50 µL of NADPH working solution to each well of NADPH standard, blank control, and test samples to make the total NADPH assay volume of 100 µL/well. For a 384-well plate, use 25 µL of working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at room temperature for 15 minutes to 2 hours, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by absorbance microplate reader at the wavelength of 576 ± 5 nm. However, the absorption detection will have a lower sensitivity compared to fluorescence reading. For cell based NADPH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (Cat No. 20012) is recommended to use for lysing the cells.

Citations


View all 64 citations: Citation Explorer
A mechanistic insight into the bioaccesible herbometallic nanodrug as potential dual therapeutic agent
Authors: Bera, Debbethi and Pal, Kunal and Ruidas, Bhuban and Mondal, Dheeraj and Pal, Shinjini and Paul, Biplab Kumar and Karmakar, Parimal and Das, Sukhen and Nandy, Papiya
Journal: Materials Today Communications (2020): 101099
Guar gum micro-vehicle mediated delivery strategy and synergistic activity of thymoquinone and piperine: An in vitro study on bacterial and hepatocellular carcinoma cells
Authors: Das, Sanghita and Bera, Debbethi and Pal, Kunal and Mondal, Dheeraj and Karmakar, Parimal and Das, Sukhen and Dey, Anindita
Journal: Journal of Drug Delivery Science and Technology (2020): 101994
Safflor Yellow B Attenuates Ischemic Brain Injury via Downregulation of Long Noncoding AK046177 and Inhibition of MicroRNA-134 Expression in Rats
Authors: Wang, Chaoyun and Wan, Hongzhi and Wang, Qiaoyun and Sun, Hongliu and Sun, Yeying and Wang, Kexin and Zhang, Chunxiang
Journal: Oxidative Medicine and Cellular Longevity (2020)
Herbometallic nano-drug inducing metastatic growth inhibition in breast cancer through intracellular energy depletion.
Authors: Ruidas, Bhuban and Sur, Tapas Kumar and Pal, Kunal and Som Chaudhury, Sutapa and Prasad, Parash and Sinha, Koel and Sarkar, Prasanta Kumar and Das, Pritha and Das Mukhopadhyay, Chitrangada
Journal: Molecular biology reports (2020): 3745--3763
Resveratrol attenuates excessive ethanol exposure induced insulin resistance in rats via improving NAD+/NADH ratio
Authors: Luo, Gang and Huang, Bingqing and Qiu, Xiang and Xiao, Lin and Wang, Ning and Gao, Qin and Yang, Wei and Hao, Liping
Journal: Molecular Nutrition & Food Research (2017)
Epigenetic regulation of Runx2 transcription and osteoblast differentiation by nicotinamide phosphoribosyltransferase
Authors: Ling, Min and Huang, Peixin and Islam, Shamima and Heruth, Daniel P and Li, Xuanan and Zhang, Li Qin and Li, Ding-You and Hu, Zhaohui and Ye, Shui Qing
Journal: Cell & Bioscience (2017): 27
MCU-dependent mitochondrial Ca2+ inhibits NAD+/SIRT3/SOD2 pathway to promote ROS production and metastasis of HCC cells
Authors: Ren, T and Zhang, H and Wang, J and Zhu, J and Jin, M and Wu, Y and Guo, X and Ji, L and Huang, Q and Yang, H and others, undefined
Journal: Oncogene (2017)
Metabolic and molecular insights into an essential role of nicotinamide phosphoribosyltransferase
Authors: Zhang, Li Q and Van Ha, undefined and el, Leon and Xiong, Min and Huang, Peixin and Heruth, Daniel P and Bi, Charlie and Gaedigk, Roger and Jiang, Xun and Li, Ding-You and Wyckoff, Gerald and others, undefined
Journal: Cell Death & Disease (2017): e2705
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

References


View all 1 references: Citation Explorer
Inhibition of leucine aminopeptidase 3 suppresses invasion of ovarian cancer cells through down-regulation of fascin and MMP-2/9
Authors: Wang X, Shi L, Deng Y, Qu M, Mao S, Xu L, Xu W, Fang C.
Journal: Eur J Pharmacol (2015): 116