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NAD+/NADH Assay Kit with WST-8

NAD+/NADH Assay Kit with WST-8

€ 302,00

Beyotime’s NAD+/NADH Assay Kit with WST-8 provides a simple colorimetric method for detecting the content of NAD+ (oxidative coenzyme I) and NADH (reductive coenzyme I) in cells, tissues or other samples, employing the WST-8 chromogenic substrate.

Package: 100T

SKU: S0175
Category: , , ,

Description

Beyotime’s NAD+/NADH Assay Kit with WST-8 provides a simple colorimetric method for detecting the content of NAD+ (oxidative coenzyme I) and NADH (reductive coenzyme I) in cells, tissues or other samples, employing the WST-8 chromogenic substrate.

The traditional method for detecting NADP+/NADPH and NAD+/NADH is to detect the absorption change of NADPH or NADH at 340nm, which has low sensitivity and is susceptible to the interference of substances with absorbance under UV light.

WST-8 is an upgraded alternative to MTT, with significant advantages over MTT, XTT and MTS. Firstly, compared to insoluble formazan produced from MTT and the low solubility of formazan from XTT and MTS, the formazan produced from WST has the highest solubility, which is of particularly importance for subsequent analysis. Secondly, WST-8 is more stable than XTT and MTS, generating more robust assays. Finally, WST-8 demonstrates a wider linear range and higher sensitivity compared to MTT and XTT.

This product is convenient to use. It does not require the purification of NAD+ and NADH from samples, and specifically detects NAD+ and NADH, but not NADP+ nor NADPH. This product is also sensitive and can detect NAD+ or NADH down to 0.25μM (5pmol), showing a good linearity from 0.25μM (5pmol) to 10μM (200pmol).

Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all cells and includes both NAD+ (oxidized) and NADH (reduced) forms. NAD+ is both a coenzyme that transfers electrons during redox reactions and a substrate for many enzymes involved in intracellular reactions. For example, deacetylases such as Sirt1 require NAD+ as a substrate in deacetylation reactions to regulate the acetylation level of proteins. NAD+ plays important roles in cells. Its synthesis and degradation and its products are involved in apoptosis, metabolic regulation and regulation of gene expression, and the decrease of NAD+ is one of the main factors of cell death. The importance of NAD+ in regulating cellular redox, signaling pathways and transcription makes NAD+ and related enzymes potential drug targets for a variety of diseases.

This product can be applied to analyze the content of total NAD, NAD+, NADH, and the ratio of NAD+/NADH. The detection principle of this kit is as below:
a. Measurement of total NAD: Ethanol is oxidized into Acetaldehyde by Alcohol dehydrogenase (ADH), during which NAD+ is reduced to NADH. In the presence of the electron coupling reagent 1-mPMS (1-Methoxy-5-methylphenazinium Methyl Sulfate), the generated NADH reduces WST-8 to an orange water soluble formazan with a maximum absorption at 450nm. The formazan formed in the reaction mixture is proportional to the total NAD+/NADH in the sample. The principle of WST-8 method for the detection of total NAD is shown in Figure 1.
b. Measurement of NADH only: After incubating samples at 60℃ for 30 minutes, NAD+ in samples is degraded while NADH remains. Therefore, the formazan generated in the reaction mixture represents the content of NADH only.
c. Determination of NAD+/NADH ratio: The ratio of NAD+/NADH can be calculated based on the amount of total NAD and NADH obtained above .

Figure 1. The detection principle of Beyotime’s NAD+/NADH Assay Kit with WST-8 (S0175).

This kit is sufficient for 100 assays when analyzing the total content of NAD or the content of NADH in 96-well plates, while it is sufficient for 50 assays when analyzing the content of NAD+ or the ratio of NAD+/NADH in 96-well plates.

Packing List：
Item Component Quantity
S0175-1 Alcohol dehydrogenase 220μl
S0175-2 Developer Solution 1.1ml
S0175-3 NADH 5mg
S0175-4 NADH Dilution Buffer 0.8ml
S0175-5 NAD+/NADH Extraction Buffer 60ml
S0175-6 Assay Buffer 10ml
Manual – 1 copy

 

Storage Conditions：

Store at -20℃ for up to 1 year. The Developer Solution and NADH are light sensitive and should be kept in the dark. NADH after reconstitution should be aliquoted and kept at -80℃. All reagents should be avoided from repeated freeze-thaws.

NADH in solution is not very stable. Use it immediately after thawing. The abnormal standard curve of NADH could be due to the degradation of NADH.

Precautions：

All reagents in the kit should be kept frozen as stated in the Storage Conditions. Make aliquots to avoid repeated freeze-thaws if the reagent could not be used up at one time.

The NAD+/NADH Extraction Buffer is viscous. When it is used for dilution of standards or samples, the diluted solution must be mixed well to ensure reliable and consistent assay results.

When adding or mixing samples, avoid the formation of air bubbles that may interfere with the absorbance measurement.

A new standard curve must be set up each time the assay is run.

Adjust the amount of sample to be extracted or Extraction Buffer appropriately when the concentration of NAD+ or NADH is out of the linear detection range of this kit.

We recommend performing the assay with freshly prepared samples because NAD+ and NADH are unstable and easily degraded during freezing. .

This product is for R&D only. Not for drug, household, or other uses.

For your safety and health, please wear a lab coat and disposable gloves during the operation.

Additional information

Weight 1 kg
Dimensions 20 × 20 × 30 cm

Instruction

Instructions for Use：

All samples and standards should be run in duplicate.
1.Sample preparation
a. Preparation of cell samples:
For 1×106 adherent cells per well of 6-well plates, remove the culture medium, add 200μl of ice-cold NAD+/NADH Extraction Buffer, and pipette gently to promote cell lysis on ice or at room temperature.
For suspension cells, take 1×106 cells, centrifuge at 600×g for 5 minutes, remove the supernatant, add 200μl of ice-cold NAD+/NADH Extraction Buffer, and pipette gently to promote cell lysis on ice or at room temperature.
Centrifuge at 12,000×g for 5-10 minutes at 4 ℃. Take the supernatant and keep on ice for subsequent assay.
b. Preparation of tissue samples: Take 10-30mg of tissues and wash with ice-cold PBS buffer. Cut into small pieces, add 400μl of ice-cold NAD+/NADH Extraction Buffer, and disrupt cells with a homogenizer on ice or at room temperature. Centrifuge at 12,000×g for 5-10 minutes at 4 ℃. Take the supernatant and keep on ice for subsequent assay.
2.Reagent preparation
a. NAPH Standard preparation: Dissolve the NADH powder (5mg) provided in the kit with 655μl of NADH Dilution Buffer to make 10mM NADH standard solution. Keep aliquots at -80℃ in the dark.
b. Preparation of NADH standard curve: Dilute the 10mM NADH standard solution with the NAD+/NADH Extraction Buffer to make the concentrations of 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 10μM. Add 20μl of each dilution into 96-well plates, with 0, 5, 10, 20, 40, 80, 120, 160, or 200pmol NADH per well, respectively. The concentrations of NADH for plotting the standard curve can be adjusted based on the NADH content in samples.
Note: NADH is unstable and must be used immediately after preparation.
c. Preparation of alcohol dehydrogenase working solution: Prepare an appropriate amount of alcohol dehydrogenase working solution by diluting the Alcohol Dehydrogenase provided in the kit with the Assay Buffer at a ratio of 1:44 (e.g., dilute 2μl of alcohol dehydrogenase with 88μl of Assay Buffer). Each assay requires 90μl of alcohol dehydrogenase working solution.
Note: The alcohol dehydrogenase working solution should be prepared freshly.
3.Assay protocol
a. Measurement of total NAD (NAD+ and NADH): Add 20μl of sample supernatant prepared in step 1 into 96-well plates. When the content of NAD in a sample is out of the linear range of the standard curve, repeat the assay after diluting the sample with the NAD+ /NADH Extraction Buffer when the NAD content is too high or using more cells or tissues for NAD extraction when the NAD content is too low in the sample.
b. Measurement of NAD+, NADH or NAD+/NADH ratio: Pipette 50-100μl of supernatant prepared in step 1 into a centrifuge tube, incubate for 30 minutes at 60℃ in a water bath or a PCR thermocycler to degrade NAD+. If precipitate forms after incubation, centrifuge at 10,000×g for 5 minutes at 4℃ or room temperature, and pipette 20μl of supernatant into 96-well plates. When the content of NAD+ or NADH in a sample is out of the linear range of the standard curve, repeat the assay after diluting the sample with the NAD+ /NADH Extraction Buffer when the NAD+ or NADH content is too high or using more cells or tissues for NAD extraction when the NAD+ or NADH content is too low in the sample.
c. Set up reactions in 96-well plates as indicated in the table. Mix well after adding alcohol dehydrogenase working solution and avoid the formation of air bubbles.

Blank Standard Sample
Sample – 20μl 20μl
NAD+/NADH Extraction Buffer 20μl – –
Alcohol Dehydrogenase Working Solution 90μl 90μl 90μl

d. Incubate at 37℃ for 10 minutes in the dark. The NAD+ will be transformed into NADH at this step.
e. Add 10μl of Developer Solution per well, mix well, and incubate at 37℃ for 30 minutes in the dark.
f. Measure the absorbance at 450nm.  The incubation time can be extended to 45-60 minutes if the A450 value is too small.
4.Calculation of NAD+/NADH content in samples
a. Correct the A450 of NADH standards and samples by subtracting the A450 of blank control from the A450 of NADH standards and samples.
b. Plot the NADH standard curve with the NADH concentrations on the x-axis and their corresponding corrected A450 on the y-axis (Figure 2).

Figure 2. Standard curves of NAD+ and NADH obtained using Beyotime’s NAD+/NADH Assay Kit with WST-8 (S0175). This figure demonstrates there is a good linear relationship between the A450 and the NAD+/NADH content in samples and this kit specifically detects NAD+ and NADH, but not NADPH. This figure is for reference only, which may vary due to different experimental conditions.
c. Determine the NAD+/NADH concentration of a sample based on its corrected A450 from the standard curve. The total content of NAD (NADtotal) including NAD+ and NADH in a sample is obtained from the reaction without 60℃ incubation, while the NADH content is obtained if the reaction is incubated at  60℃.
d. The NAD+ content and NAD+/NADH ratio in a sample can be obtained using the formula below. At this time, the NAD+ and NADH content can be reported as pmol/cell number or pmol/mg tissue weight.
[NAD+] = [NADtotal] – [NADH]
[NAD+]/[NADH] = ([NADtotal] – [NADH])/[NADH]
e. If an absolute quantification of NAD+ and NADH in a sample is desired, determine the protein concentration of the sample with the BCA Protein Assay method and then report the NAD+ and NADH content as μmol/mg protein.

Product files

MSDS

https://scandicbiotech.com/wp-content/uploads/2026/04/S0175.pdf

References

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