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| Product Name | NAD/NADH Quantification Colorimetric Assay Kit |
| Catalog No. | CMTR-HMM-0055 |
| Description | A colorimetric assay for the separate measurement of total (NAD+ + NADH) nicotinamide adenine dinucleotide and NADH alone in biological samples. This enables calculation of the NAD+/NADH ratio, a key indicator of cellular redox state and metabolic status. The assay employs an alcohol dehydrogenase cycling reaction to amplify the signal for increased sensitivity. |
| Intended Use | Determination of intracellular NAD+ and NADH concentrations and the NAD+/NADH ratio in cells and tissues under different metabolic conditions for research in mitochondrial function, oxidative stress, aging, cancer metabolism, and metabolic disease. |
| Principle / Technology | NAD+ and NADH are extracted from samples under appropriate conditions (acid extraction for NAD+ or alkaline/heat extraction for NADH); in the cycling enzyme reaction, alcohol dehydrogenase catalyzes the reduction of NAD+ to NADH using ethanol as substrate; the generated NADH then reduces a tetrazolium salt to a colored formazan product via diaphorase, generating a signal that cycles and amplifies up to 100-fold for picomolar-level quantification. |
| Detection Method | Colorimetric absorbance measurement at 450 nm; absorbance at 570 nm for the formazan product with optional reference at 650 nm using a microplate reader |
| Sample Type | Cultured mammalian cells, fresh or frozen tissue samples, isolated mitochondria, blood cells, yeast and bacterial cell extracts |
| Performance Range / Specifications | NAD(H) quantification range: 0.5–50 pmol/well in 96-well plate format; standard curve range: 0–10 μM NADH; total NAD and NADH separately measured in parallel plates or sequential assays; NAD+/NADH ratio typically 3–10 in healthy proliferating cells |
| Sensitivity / LOD | Detection limit: approximately 0.1 μM NADH in sample extract at a 1:10 dilution factor; measurable in as few as 2,000 cells per well for dividing mammalian cells |
| Specificity | The NAD/NADH cycling reaction is specific for nicotinamide adenine dinucleotides; nicotinamide adenine dinucleotide phosphate (NADP/NADPH) is not recognized by alcohol dehydrogenase in the cycling enzyme mix; separate extraction procedures ensure selective measurement of total NAD and NADH levels |
| Reaction Conditions / Protocol | Prepare cells or tissue: wash with cold PBS; for total NAD measurement, lyse cells with acid extraction buffer and neutralize; for NADH measurement, lyse with alkaline extraction buffer and heat at 60°C for 30 minutes (NAD+ is decomposed under these conditions); add extracted samples and NADH standards to plate; add Cycling Enzyme Mix; incubate 1–4 hours at room temperature; measure absorbance at 450 nm |
| Components / Formulation | NADH Standard (lyophilized, for standard curve), NAD Extraction Buffer (acidic), NADH Extraction Buffer (alkaline), Cycling Buffer, Cycling Enzyme Mix (alcohol dehydrogenase, diaphorase, ethanol, WST-1 tetrazolium salt), Neutralization Buffer, detailed technical manual |
| Storage Conditions | Lyophilized and concentrated solutions at -20°C; buffers at 2–8°C; reconstituted NADH stable for 1 week at -20°C; enzyme mix sensitive to freeze-thaw (aliquot upon first use) |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 100 assays, 400 assays (96-well format) |
| Product Form | Lyophilized enzymes and standards with liquid extraction buffers |
| Quality Control | Each lot verified for NADH standard curve linearity (R2 ≥0.995); sensitivity confirmed by quantification of known NADH spike into cell lysate; lot-to-lot consistency within 15% |
| Key Features | Separate measurement protocols for total NAD and NADH allow accurate redox ratio calculation; enzyme cycling amplification enhances signal for low-abundance samples; compatible with tissue and cell samples across species; NADH standard curve included for absolute quantification |
For research use only, not for clinical use.
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