| Field | Specification |
|---|---|
| Assay Time | |
| Detection Method | |
| Product Type | |
| Sample Type(s) | Cell, tissue extracts etc Direct NAD/NADH Assays in 96-Well Plate NEW!!! |
| Shipping | |
| Species | |
| Storage |
Overview
For sensitive determination of NAD and NADH and evaluation of drug effects on NAD/NADH metabolism. The assay uses F530/585nm for signal readout. Compatible sample input includes Cell, tissue extracts etc Direct NAD/NADH Assays in 96-Well Plate NEW!!!. Typical stated assay timing is 10 min.
Key elements and design rationale
- Readout format: F530/585nm supports plate-based signal acquisition and consistent comparison across matched samples.
- Sample compatibility: The stated sample scope includes Cell, tissue extracts etc Direct NAD/NADH Assays in 96-Well Plate NEW!!!, which is useful when aligning matrix type with calibration and control design.
- Analytical range context: The supplied specifications include a stated detection limit of 0.02 µM for interpreting low-signal samples.
- Feature emphasis: Sensitive and accurate. Detection limit of 0.02 µM and linearity up to 1 µM NAD+/NADH in 96-well plate assay.
Additional feature notes highlight Convenient. The procedure involves adding a single working reagent, and reading the fluorescence at time zero and 10 min; High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day. Available format information for this listing includes 100 Tests.
Biological background
This product is centered on measurement of enzyfluo nad/nadh within the matrices described for the assay. In practice, datasets from this type of format are typically interpreted by comparing relative signal, activity, or abundance across matched control and experimental groups rather than relying on a single value in isolation. Careful alignment of sample matrix, incubation window, and calibration strategy is important when comparing results across plates, operators, or study days.
More details
Pyridine nucleotides play an important role in metabolism and, thus, there is continual interest in monitoring their concentration levels. Quantitative determination of NAD+/NADH has applications in research pertaining to energy transformation and redox state of cells or tissue. BioAssay Systems EnzyFluo™ NAD+/NADH assay kit is based on a lactate dehydrogenase cycling reaction, in which the formed NADH reduces a probe into a highly fluorescent product. The fluorescence intensity of this product, measured at λex/em = 530/585 nm, is proportional to the NAD+/NADH concentration in the sample. This assay is highly specific for NAD+/NADH with minimal interference (<1%) by NADP+/NADPH and is a convenient method to measure NAD, NADH and their ratio.Direct NAD/NADH Assays in 96-Well PlateNEW!!!
Detection method
Fluorescent (F530/585nm).
Detection limit and analytical sensitivity
Reported detection limit: 0.02 µM.
Procedures and timing
Stated procedure or timing information: 10 min.
Research relevance and current trends
- Plate-based quantification and side-by-side group comparison remain central use cases for this assay format.
- The product notes emphasize multi-sample throughput, making it relevant for screening-oriented and larger batch comparison studies.
- The description supports intervention-focused study designs in which researchers compare baseline and perturbed conditions.
Common research applications
- Quantify enzyfluo nad/nadh in cell, tissue extracts Direct NAD/NADH Assays by F530/585 nm readout.
- Compare treatment or phenotype groups using matched cell, tissue extracts Direct NAD/NADH Assays handling.
- Monitor time-course or pre/post changes in cell, tissue extracts Direct NAD/NADH Assays across study conditions.
Interpretation is usually strongest when signal changes are assessed alongside matrix-matched controls, replicate agreement, and the assay's stated analytical window.
Notes for experimental interpretation
- Matrix composition, background signal, and sample handling can influence apparent response; compare like-with-like whenever possible.
- Use appropriate blanks, controls, and replicate wells to distinguish biological differences from plate, reagent, or handling variability.
Can I determine NAD/NADH directly in 96-well plates?
Yes, NAD/NADH can be determined directly in 96-well plate. Please see our optimized protocol Direct NAD/NADH Assays in 96-Well Plate
Direct NAD/NADH Assays in 96-Well Plate
When tissue is used, should it be freshly obtained? Or is -80°C storage ok?
If direct sample processing is not possible, we recommend snap freezing tissue samples in liquid nitrogen and keeping them either in liquid nitrogen or at -80°C until further processing.
I have conducted protocols that involve trypsinizing cells, washing with PBS, pelleting, and then applying extraction buffer. My concern with this method is that PBS is known to cause mitochondrial fragmentation very soon after application to cells. Obviously, the functionality of the mitochondria is very important for NADH/NAD levels. I developed an alternative by keeping my cells adhered to the plate, washing once with pbs, applying heated extraction buffer to the cells directly and then scraping cells off. Do you have any thoughts or experience that would indicate whether one method is better than the other?
The second method is better because the first method may cause mitochondrial fragmentation and thus may alter intracellular NAD/NADH concentrations. The second method involves less pretreatment. Adding the heated extraction buffer terminates the metabolic reactions in the cells and is thus much less likely to alter the intracellular NAD/NADH concentrations.
I have also done many experiments with different numbers of cells. What I notice is that there seems to be a sigmoidal relationship between the number of cells and the NAD and NADH concentrations. I am using C2C12 and H9C2 cells. Do you have any experience with these cells at different seeding densities? Also, do you consistently see a relationship between number of cells NAD(H) concentrations? Is this relationship linear, exponential, sigmoidal?
We have not done these experiments. Your results are very interesting, and not unexpected. In general, cells are healthy at certain densities. When cells lack contacts for a healthy environment at low density, they are likely to produce less NAD/NADH, and if too dense, the cells become less active and thus would reach a plateau in NAD/NADH concentrations.
For laboratories requiring additional technical capacity, we provide scientific support services including assay execution, method guidance, product sourcing, and customization to align the assay with specific experimental objectives. If you need assistance selecting the appropriate kit configuration, adapting the workflow to your application, or identifying related research services, please click Talk to a Scientist, email support@biohippo.com, or review our Research Services; a member of our scientific team will follow up with recommendations tailored to your study.
Pathogen induced subversion of NAD+ metabolism mediating host cell death: a target for development of chemotherapeutics
Chaurasiya, A., et al. (2021). Pathogen induced subversion of NAD+ metabolism mediating host cell death: a target for development of chemotherapeutics. Cell Death Discovery 7(1): 10. Assay: NAD and NADH in microbial cells.
Cross-talk between CD38 and TTP is essential for resolution of inflammation during microbial sepsis
Joe, Y., et al. (2020). Cross-talk between CD38 and TTP is essential for resolution of inflammation during microbial sepsis. Cell Reports 30(4): 1063-1076.e5. Assay: NAD and NADH in mouse cells.
Dysregulated metabolic pathways in age-related macular degeneration
Zhang, M., et al. (2020). Dysregulated metabolic pathways in age-related macular degeneration. Scientific Reports 10(1): 2464. Assay: NAD and NADH in human cells.
Acute stimulation of glucose influx upon mitoenergetic dysfunction requires LKB1, AMPK, Sirt2 and mTOR-RAPTOR
Liemburg-Apers, Dania C., et al (2016). Acute stimulation of glucose influx upon mitoenergetic dysfunction requires LKB1, AMPK, Sirt2 and mTOR-RAPTOR. J Cell Sci 129.23: 4411-4423. Assay: NAD/NADH in mouse cells.
Kim, Ha-Neui, et al. (2015) Sirtuin1 suppresses osteoclastogenesis by deacetylating FoxOs. Molecular Endocrinology 29.10
Kim, Ha-Neui, et al. (2015) Sirtuin1 suppresses osteoclastogenesis by deacetylating FoxOs. Molecular Endocrinology 29.10: 1498-1509. Assay: NAD/NADH in mice.
Research budgets are tight — we get it. That's why we've put together a fresh round of exclusive promotions designed to help you stock up on the reagents, kits, and consumables your lab depends on, without stretching your budget.
🔬 What's on offer right now:
10% Off Pre-Designed siRNA Sets
20% Off Transmembrane Proteins
50% Off Lab Consumables + Free Shipping
$99 Pipette Filler Promotion Package
BlasTaq 2X qPCR MasterMix - 50% OFF Limited Time Offer
DENARASE® Endonuclease — 10% Off One Order
