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| Product Name | NAD/NADH Ratio Fluorometric Assay Kit, Cyclic Enzyme Method |
| Catalog No. | CATR-HMM-0061 |
| Description | Fluorometric assay kit for quantitative measurement of intracellular nicotinamide adenine dinucleotide (NAD+ and NADH) levels and the NAD+/NADH ratio in cell and tissue extracts. The assay employs a cyclic enzyme amplification system where NAD+/NADH is continuously recycled by alcohol dehydrogenase and diaphorase reactions, generating a fluorescent resorufin product (Ex/Em 540/590 nm) proportional to the total NAD (NAD+ + NADH) or NADH-only in the sample. Separate extraction protocols for NAD+ (acid extraction) and NADH (alkaline extraction) enable differential quantification of both forms from the same sample. |
| Intended Use | Measurement of intracellular NAD+ and NADH levels to assess cellular redox state, energy metabolism, sirtuin activity, PARP activation, and metabolic dysfunction in cancer, aging, neurodegenerative disease, and metabolic disorder research. |
| Principle / Technology | Enzymatic cycling: alcohol dehydrogenase reduces NAD+ to NADH using ethanol; diaphorase oxidizes NADH back to NAD+ while reducing non-fluorescent resazurin to fluorescent resorufin; each NAD+/NADH molecule undergoes thousands of cycles per minute, amplifying signal >100-fold. |
| Detection Method | Fluorescence microplate reader (Ex/Em 540/590 nm); endpoint reading after 30-minute incubation at room temperature. |
| Sample Type | Cultured cell lysates (1×10^4-5×10^6 cells); fresh and frozen tissue homogenates; isolated mitochondria. |
| Performance Range / Specifications | Linear detection range: 0.01-1 µM NAD+ or NADH in the reaction well; applicable to 0.1-100 pmol per well; NAD+/NADH ratio range 0.1-10 measurable with <15% CV. |
| Sensitivity / LOD | Detection limit 0.5 nM NAD+ or NADH in the assay well (10 fmol); signal-to-background >20:1 at 10 nM. |
| Specificity | Specific for β-NAD+ and β-NADH; <0.1% cross-reactivity with NADP+/NADPH due to alcohol dehydrogenase substrate specificity. |
| Reaction Conditions / Protocol | Extract NAD+ (acid lysis) and NADH (alkaline lysis + heat 60 °C, 30 min); neutralize extracts; mix with cycling enzyme reagent; incubate 30 min at RT; read fluorescence Ex/Em 540/590 nm. |
| Components / Formulation | NAD+/NADH extraction buffers (acid and alkaline), neutralizing buffer, NADH standard (1 µmol), NAD cycling enzyme mix (lyophilized), NADH cycling enzyme mix (lyophilized), cycling buffer, resazurin probe, 96-well black microplate. |
| Storage Conditions | Store enzyme mixes and probe at -20 °C; extraction buffers at 2-8 °C; NADH standard at -20 °C protect from light and moisture. |
| Shelf Life | 6 months from date of manufacture. |
| Package Specifications | 100 tests, 400 tests (based on duplicate wells for both NAD+ and NADH measurements). |
| Product Form | Lyophilized enzyme mixes and probe; liquid extraction buffers; white powder NADH standard. |
| Quality Control | Each lot tested for cycling efficiency using 0.1 µM NADH standard; fluorescence signal linearity across operating range R² ≥0.995; enzyme activity verified. |
| Key Features | Enzymatic cycling amplification (1000-fold signal enhancement); separate extraction for NAD+ and NADH from same sample; high sensitivity (10 fmol detection limit); NADP+/NADPH interference <0.1%; compatible with tissue and cell samples. |
| Purity | Enzyme mixes contain purified recombinant proteins; resazurin probe ≥98% purity. |
| Concentration | NADH standard: prepare 10 µM working solution in extraction buffer. |
| Activity / Unit Definition | Alcohol dehydrogenase specific activity ≥300 U/mg; diaphorase specific activity ≥100 U/mg. |
| Molecular Weight | NAD+: 663.43 g/mol; NADH: 665.44 g/mol. |
| Source / Origin | Recombinant alcohol dehydrogenase (S. cerevisiae) and diaphorase (C. kluyveri) expressed in E. coli; synthetic resazurin. |
| pH Range / Optimal pH | Extraction pH: NAD+ (pH 2-3 acid), NADH (pH 12-13 alkaline); cycling assay pH 7.5-8.0. |
| Shipping Conditions | Cold pack (enzyme mixes and standard on dry ice recommended). |
| Expiration Date / Stability | 6 months at recommended storage; reconstituted enzyme mixes stable for 2 weeks at 2-8 °C. |
| Regulatory / Compliance | For research use only; not for diagnostic or clinical applications. |
| Compatibility | Compatible with cell and tissue extracts prepared using provided extraction buffers. Detergents >0.1% may inhibit enzyme cycling; use recommended extraction protocol. Hemoglobin and bilirubin in tissue lysates may quench fluorescence. |
| Recommended Buffer System | Cycling buffer: Tris-HCl 100 mM, pH 8.0, ethanol 2% v/v. |
| Application Notes / Precautions | Work quickly during the extraction steps as NADH is unstable at acidic pH and degrades within minutes. Keep all samples and reagents on ice during extraction. Normalize NAD+/NADH values to protein content for accurate comparison between samples. Acid extraction for NAD+ destroys NADH — ensure complete separation of aliquots before adding extraction buffers. Use black plates with clear bottoms for optimal fluorescence measurement. |
| Batch-to-Batch Consistency | Cycling enzyme activity within ±15% of reference lot; NADH standard concentration verified by absorbance at 340 nm (ε = 6.22 mM-1cm-1). |
For research use only, not for clinical use.
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