NAD+/NADH Colorimetric Assay Kit (WST-8)
NAD+/NADH Colorimetric Assay Kit (WST-8)
| SKU # | E-BC-K804-M |
| Detection Instrument | Microplate reader (450 nm) |
| Detection method | Colorimetric method |
Product Details
Properties
| Synonyms | NAD+/NADH |
| Sample type | Animal tissue, cells |
| Sensitivity | 0.02 μmol/L |
| Detection range | 0.02-5.0 μmol/L |
| Detection method | Colorimetric method |
| Assay type | Quantitative |
| Assay time | 70 min |
| Precision | Average inter-assay CV: 9.100% | Average intra-assay CV: 1.800% |
| Other instruments required | Micropipettor, 37℃ water bath, 10 KD filters tube |
| Other reagents required | PBS(0.01 M, pH 7.4) |
| Storage | -20℃ |
| Valid period | 12 months |
Dilution of Sample
The recommended dilution factor for different samples is as follows (for reference only):
| Sample type | Dilution factor |
| 10% Mouse muscle tissue homogenate | 1 |
| 10% Mouse kidney tissue homogenate | 1 |
| 293T cell | 1 |
| Hela cell | 1 |
Note: The diluent is extracting solution. For the dilution of other sample types, please do pretest to confirm the dilution factor.
Detection Principle
Detect total content of NAD+ and NADH Ethanol generates acetaldehyde under the action of enzyme. Meanwhile, NAD+ is reduced to NADH, NADH, under the action of hydrogen transmitter, transfer electrons to WST-8 to produce the yellow product, which has a characteristic absorption peak at 450 nm. Therefore, the total content of NAD+ and NADH can be quantified by measure the OD value at 450 nm. Detect NADH After treating sample, heat at 60oC water bath for 30 min. the NAD+ of the sample is decomposed and only NADH remains. NADH reduces WST-8 to form yellow product, and the amount of NADH is determined by measure the OD value at 450 nm. Detect NAD+ and NAD+/NADH The content of NAD+ and the ratio of NAD+/NADH in the sample can be obtained according to the total content of NAD+ and NADH obtained of the first two steps as well as the separate content of NADPH. Note: NADP+ and NADPH have no effect on the determination results.
Kit Components & Storage
| Item | Component | Size (96 T) | Storage |
| Reagent 1 | Extracting Solution | 60 mL ×2 vials | -20℃, 12 months |
| Reagent 2 | Buffer Solution | 16 mL × 1 vial | -20℃, 12 months |
| Reagent 3 | Chromogenic Agent | 5 mL × 1 vial | -20℃, 12 months, shading light |
| Reagent 4 | Enzyme Reagent | Powder × 2 vials | -20℃, 12 months, shading light |
| Reagent 5 | Standard | Powder × 2 vials | -20℃, 12 months, shading light |
| Microplate | 96 wells | No requirement | |
| Plate Sealer | 2 pieces |
Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as not to obtain sufficient amount of reagents.
Technical Data:
Parameter:
Intra-assay Precision
Three mouse kidney tissue samples were assayed in replicates of 20 to determine precision within an assay (CV = Coefficient of Variation).
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (μmol/L) | 0.55 | 2.10 | 4.30 |
| %CV | 2.3 | 1.7 | 1.4 |
Inter-assay Precision
Three mouse kidney tissue samples were assayed 20 times in duplicate by three operators to determine precision between assays.
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (μmol/L) | 0.55 | 2.10 | 4.30 |
| %CV | 8.5 | 9.2 | 9.6 |
Recovery
Take three samples of high concentration, middle concentration and low concentration to test the samples of each concentration for 6 times parallelly to get the average recovery rate of 90%.
| Standard 1 | Standard 2 | Standard 3 | |
| Expected Conc. (μmol/L) | 1.2 | 2.4 | 3.8 |
| Observed Conc. (μmol/L) | 1.1 | 2.2 | 3.4 |
| Recovery rate(%) | 89 | 92 | 89 |
Sensitivity
The analytical sensitivity of the assay is 0.02 μmol/L. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.
Standard Curve
As the OD value of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique or temperature effects), so the standard curve and data are provided as below for reference only:
| Concentration (μmol/L) | 0 | 1.0 | 1.5 | 2.0 | 2.5 | 3.0 | 4.0 | 5.0 |
| Average OD | 0.153 | 0.454 | 0.584 | 0.724 | 0.854 | 1.103 | 1.290 | 1.550 |
| Absoluted OD | 0.000 | 0.301 | 0.431 | 0.571 | 0.701 | 0.950 | 1.137 | 1.397 |