NADP+/NADPH Assay Kit (WST-8)
Based on 2 publication(s) in Google Scholar
MCE NADP+/NADPH Assay Kit (WST-8) is a colorimetric assay that conveniently detects the oxidized (NADP+) and reduced (NADPH) forms of coenzyme II within cells, tissues, and other samples.
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Storage :
-20℃, 1 year
Keep away from light and avoid repeat freeze-thaw cycles
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Description & Advantages
Nicotinamide Adenine Dinucleotide Phosphate (NADP) serves as a coenzyme in many redox reactions, existing in both oxidized (NADP+) and reduced (NADPH) forms.
MCE NADP+/NADPH Assay Kit (WST-8) is a colorimetric assay that conveniently detects the oxidized (NADP+) and reduced (NADPH) forms of coenzyme II within cells, tissues, and other samples. This is achieved through colorimetric measurements based on the WST-8 color reaction, eliminating the need for purification of NADP+/NADPH from the samples.
Protocol
1. Sample extraction
Add an appropriate volume of NADP+/NADPH Extract Solution to cells or tissues, homogenize/lyse thoroughly, centrifuge, and collect the supernatant. Perform extraction on ice whenever possible.
2. NADPH standard curve
Prepare an NADPH stock from the supplied standard and serially dilute with Extract Solution:
| Standard | NADPH standard | NADP+/NADPH Extract Solution | NADPH concentration |
|---|---|---|---|
| S0 | 0 μL | Bring to final volume | 0 |
| S1-S5 | Serially diluted standard | Bring to equal final volume | According to dilution series |
3. NADPH and total NADP(H) samples
Use one aliquot of the sample directly to determine total NADP+ + NADPH. Subject an equal aliquot to the selective pretreatment described in the Manual to remove NADP+ and measure NADPH. Calculate NADP+ as total NADP(H) minus NADPH.
4. WST-8 colorimetric reaction
| Component | Suggested amount per well |
|---|---|
| Reaction Buffer | 90 μL |
| G6PDH | 2 μL |
| Chromogen Solution | 10 μL |
| Standard or sample | Add as required while keeping equal final volume |
Mix and incubate at 37°C for approximately 30 min protected from light. Measure absorbance at 450 nm.
5. Calculation
Calculate NADPH and total NADP(H) from the NADPH standard curve, then calculate NADP+ = total NADP(H) - NADPH. Normalize to sample mass, cell number, or protein amount as appropriate.
Note: Protect Chromogen Solution from light and avoid repeated freeze-thaw cycles.
Publications
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Journal Impact Factor
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Most Recent
Storage
-20℃, 1 year
Keep away from light and avoid repeat freeze-thaw cycles
Components
| Components | HY-K1501-100 T |
|---|---|
| NADP+/NADPH Extract Solution | 50 mL |
| NADPH | 0.5 mg |
| G6PDH | 200 μL |
| Chromogen Solution | 1.1 mL |
| Reaction Buffer | 10 mL |
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