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NAD-Malate Dehydrogenase (NAD-MDH) Activity Assay Kit
SKU: E-BC-K561-M-96
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NAD-Malate Dehydrogenase (NAD-MDH) Activity Assay Kit
| SKU # | E-BC-K561-M |
| Detection Instrument | Microplate reader (440-460 nm, optimum wavelength: 450 nm) |
| Detection method | Colorimetric method |
Product Details
Properties
| Synonyms | NAD-MDH |
| Sample type | Serum (plasma), animal tissue, cell |
| Sensitivity | 1.06 U/L |
| Detection range | 1.06–46.7 U/L |
| Detection Method | Colorimetric method |
| Assay type | Enzyme Activity |
| Assay time | 30 min |
| Precision | Average inter-assay CV: 8.400% | Average intra-assay CV: 3% |
| Other instruments required | Incubator, centrifuge |
| 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% Rat kidney tissue homogenate | 1 |
| 10% Rat spleen tissue homogenate | 1 |
| 10% Rat liver tissue homogenate | 1 |
| 10% Rat heart tissue homogenate | 1 |
| 10% Mouse liver tissue homogenate | 1 |
| 10% Mouse lung tissue homogenate | 1 |
| Mouse serum | 1 |
| Rat serum | 1 |
| Mouse plasma | 1 |
| Rat plasma | 1 |
| Human serum | 1 |
| Dog serum | 1 |
| HT29 cell | 1 |
| Molt-4 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
NAD-MDH catalyzes the conversion of substrate malic acid and NAD to oxaloacetic acid and NADH. NADH makes WST-8 orange under the action of electron coupling agent. The activity of NAD-MDH can be calculated by measuring the change of absorbance value at 450 nm.
Kit Components & Storage
| Item | Component | Size 1(48 T) | Size 2(96 T) | Storage |
| Reagent 1 | Extracting Solution | 60 mL × 1 vial | 60 mL × 2 vials | -20°C, 12 months |
| Reagent 2 | Buffer Solution A | 10 mL × 1 vial | 20 mL × 1 vial | -20°C, 12 months |
| Reagent 3 | Buffer Solution B | 5 mL × 1 vial | 10 mL × 1 vial | -20°C, 12 months |
| Reagent 4 | Substrate | Powder ×1 vial | Powder ×1 vial | -20°C, 12 months shading light |
| Reagent 5 | Chromogenic Agent | 3 mL × 1 vial | 6 mL × 1 vial | -20°C, 12 months shading light |
| Reagent 6 | Standard | Powder × 1 vial | Powder × 2 vials | -20°C, 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 human serum 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 (U/L) | 5.6 | 28.4 | 33.5 |
| %CV | 3.6 | 2.9 | 2.5 |
Inter-assay Precision
Three human serum samples were assayed 17 times in duplicate by three operators to determine precision between assays.
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (U/L) | 5.6 | 28.4 | 33.5 |
| %CV | 8.2 | 8.3 | 8.7 |
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 96%.
| Standard 1 | Standard 2 | Standard 3 | |
| Expected Conc. (μmol/L) | 125 | 264 | 375 |
| Observed Conc. (μmol/L) | 120 | 248.2 | 367.5 |
| Recovery rate (%) | 96 | 94 | 98 |
Sensitivity
The analytical sensitivity of the assay is 1.06 U/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 | 100 | 150 | 200 | 300 | 350 | 400 | 500 |
| OD value | 0.059 | 0.215 | 0.297 | 0.390 | 0.554 | 0.647 | 0.714 | 0.856 |
| 0.059 | 0.215 | 0.289 | 0.379 | 0.543 | 0.627 | 0.721 | 0.876 | |
| Average OD | 0.059 | 0.215 | 0.293 | 0.385 | 0.549 | 0.637 | 0.718 | 0.866 |
| Absoluted OD | 0.000 | 0.156 | 0.234 | 0.326 | 0.490 | 0.578 | 0.659 | 0.807 |