Amplite® Fluorimetric Total NAD and NADH Assay Kit Red Fluorescence

Ordering information

Price
Catalog Number
Unit Size
Quantity

Add to cart

Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Overview SDS Protocol

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® NAD/NADH Assay Kit provides a convenient method for sensitive detection of NAD and NADH. The enzymes in the system specifically recognize NAD/NADH in an enzyme cycling reaction. There is no need to purify NAD/NADH from sample mix. The enzyme cycling reaction significantly increases detection sensitivity. In addition, this assay has very low background since it is run in the red visible range that significantly reduces the interference from biological samples. The assay has demonstrated high sensitivity and low interference with 570 nm excitation 590 nm emission.

Platform

Fluorescence microplate reader

Excitation	540 nm
Emission	590 nm
Cutoff	570 nm
Recommended plate	Solid black

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare NAD/NADH working solution (50 µL)
Add NADH standards or test samples (50 µL)
Incubate at room temperature for 15 minutes – 2 hours
Monitor the fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 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. NADH standard solution (1 mM):
Add 200 µL of 1X PBS buffer into the vial of NADH Standard (Component C) to make 1 mM (1 nmol/µL) NADH standard solution.

PREPARATION OF STANDARD SOLUTION

NADH standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15257

Add 10 µL of 1 mM (1 nmol/µL) NADH standard solution to 990 µL 1X PBS buffer to generate 10 µM (10 pmol/µL) NADH standard solution (NS7). Take 10 µM NADH standard solution (NS7) to perform 1:3 serial dilutions in 1X PBS buffer to get serially diluted NADH standards (NS6 - NS1). Note: Diluted NADH standard solution is unstable and should be used within 4 hours.

PREPARATION OF WORKING SOLUTION

Add 10 mL of NADH Sensor Buffer (Component B) into the bottle of NAD/NADH Recycling Enzyme Mix (Component A) and mix well to make NAD/NADH working solution. Note: This NAD/NADH working solution is enough for two 96-well plates.

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

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of NADH standards and test samples in a solid black bottom 96-well microplate. NS=NADH Standards (NS1 - NS7, 0.014 to 10 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 NADH (e.g., >100 µM, final concentration) may cause reduced fluorescence signal due to the over oxidation of NADH sensor (to a non-fluorescent product).

Well	Volume	Reagent
NS1 - NS7	50 µL	Serial Dilutions (0.014 to 10 µM)
BL	50 µL	1X PBS buffer
TS	50 µL	test sample

Prepare NADH 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.
Add 50 µL of NAD/NADH working solution to each well of NADH standard, blank control, and test samples to make the total NAD/NADH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of NAD/NADH 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 fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm). Note:The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. The absorption detection has lower sensitivity compared to fluorescence reading. Note: For NAD/NADH ratio measurements, kit 15263 is recommended. Note: For cell based NAD/NADH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (cat#20012) is recommended to use for lysing the cells.

Images

Figure 1. NADH dose response was measured with Amplite® Total NAD and NADH Assay Kit in a solid black 96-well plate using a NOVOStar microplate reader (BMG Labtech). RFU at Ex/Em = 540/590 nm.

Figure 2. Effects of AA005 on ATP production and AMPK/mTOR signaling pathway. (A) Chemical structure of AA005-fluorescein. (B) The intracellular localization of AA005. The cells were co-incubated with AA005-flu at 100 nM for 12 h, and analyzed by confocal microscopy using a mitotracker (red) to counter-stain mitochondria. (C) MTT assay of LOVO, HT29, HCT116 and HBEpiC cells upon AA005-flu at indicated concentrations for 48 h. (D) AA005 decreases the mitochondrial transmembrane potential of colon cancer cells revealed by increase in Rhodamine 123-negative cells. The cells were treated with AA005 at indicated concentration for 24 h and analyzed by Rhodamine 123 staining and flow cytometry. (E) The cells were treated with or without AA005 at indicated concentration for 24 h, NAD+/NADH ratio was measured using an Amplite^TM Colorimetric NAD/NADH Assay Kit. (F) The cells were treated with or without AA005 at indicated concentration for 24 h, and ATP content was measured using an ATP Bioluminescence Assay Kit. (G) The cells were treated with AA005 at indicated concentration and time points, lysed, and Western blot analysis was performed using indicated antibodies. Source: Graph from Identification of an Annonaceous Acetogenin Mimetic, AA005, as an AMPK Activator and Autophagy Inducer in Colon Cancer Cells by Yong-Qiang Liu et al., PLOS, Oct. 2012.

Citations

View all 66 citations: Citation Explorer

NAMPT-dependent NAD+ salvage is crucial for the decision between apoptotic and necrotic cell death under oxidative stress
Authors: Nishida, Takuto and Naguro, Isao and Ichijo, Hidenori
Journal: Cell Death Discovery (2022): 1--11

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

B cell Sirt1 deacetylates histone and non-histone proteins for epigenetic modulation of AID expression and the antibody response
Authors: Gan, Huoqun and Shen, Tian and Chupp, Daniel P and Taylor, Julia R and Sanchez, Helia N and Li, Xin and Xu, Zhenming and Zan, Hong and Casali, Paolo
Journal: Science advances (2020): eaay2793

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)

Notoginsenoside R1 attenuates high glucose-induced endothelial damage in rat retinal capillary endothelial cells by modulating the intracellular redox state
Authors: Fan, Chunlan and Qiao, Yuan and Tang, Minke
Journal: Drug design, development and therapy (2017): 3343

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)

Mitochondrial elongation-mediated glucose metabolism reprogramming is essential for tumour cell survival during energy stress
Authors: Li, J and Huang, Q and Long, X and Guo, X and Sun, X and Jin, X and Li, Z and Ren, T and Yuan, P and Huang, X and others,
Journal: Oncogene (2017): 4901--4912

In vitro assay development and HTS of small-molecule human ABAD/17$\beta$-HSD10 inhibitors as therapeutics in Alzheimer’s disease
Authors: Aitken, Laura and Baillie, Gemma and Pannifer, Andrew and Morrison, Angus and Jones, Philip S and Smith, Terry K and McElroy, Stuart P and Gunn-Moore, Frank J
Journal: SLAS DISCOVERY: Advancing Life Sciences R\&D (2017): 676--685