BioReagent BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
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Cited in 0 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
Nicotinamide adenine dinucleotide phosphate (NADP) is a coenzyme for many oxidoreductases and exists in two forms: NADP⁺ (oxidized) and NADPH (reduced). NADP⁺ is also involved in biosynthetic reactions, such as the synthesis of lipids and nucleic acids. In animal cells, the oxidative phase of the pentose phosphate pathway (PPP) is the primary source of NADPH. The measurement of NADP⁺/NADPH is mainly applied in studies related to cellular or tissue energy conversion and redox status.
Assay Principle: This assay utilizes a glucose dehydrogenase cycling reaction (which does not recognize NAD⁺/NADH). The NADPH generated in this process reduces WST-8 to produce an orange-yellow formazan dye, which has a maximum absorption peak around 450 nm. The amount of formazan generated in the reaction system is proportional to the total amount of NADP⁺ or NADPH in the sample.
Detection Range: 0.5-20 µM
Sensitivity: 0.5 µM
Applicable Samples: Animal/plant tissues, cells, bacteria, serum/plasma
| Component | 48T | 96T | Storage |
| Assay Buffer | 7.5 mL | 15 mL | 2-8℃ |
| Glucose (1M) | 1 mL | 2 mL | 2-8℃ |
| WST-8 | 350 μL | 700 μL | -20℃. Store in the dark. |
| Enhancer | 70 µL | 140 µL | -20℃. Store in the dark. |
| NADP Cycling Enzyme Mix | 70 µL | 140 µL | -20℃. Store in the dark. |
| NADPH Standard | 1EA | 1EA | -20℃. Store in the dark. |
| NADP Extraction Buffer | 7 mL | 14 mL | 2-8℃ |
| NADPH Extraction Buffer | 7 mL | 14 mL | 2-8℃ |
Please check the volume of all components before the experiment.
An extra 10% volume is provided for standard curve preparation or pre-experiments.
User-Provided Instruments and Reagents
| Type | Name | Notes |
| Instrument | Microplate Reader | Capable of measuring absorbance at 450 nm |
| Consumables | 96-well microplate | Standard transparent plate |
| Reagents | PBS (pH 7.4), Deionized Water | For washing/diluting samples |
| Others | Homogenizer (for tissue samples), Incubator, Ice Maker, Refrigerated Centrifuge, Adjustable Pipettes and Tips | Using a multichannel pipette is recommended for high-throughput assays |
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Preparation | Notes |
| Assay Buffer | Ready-to-use; equilibrate to RT before use | Store at 4°C |
| Glucose (1M) | Ready-to-use; equilibrate to RT before use | Store at 4°C |
| WST-8 | Ready-to-use | Keep on ice protected from light during experiment; store aliquoted at -20°C protected from light |
| Enhancer | Ready-to-use | Keep on ice protected from light during experiment; store aliquoted at -20°C protected from light |
| NADP Cycling Enzyme Mix | Ready-to-use | Keep on ice protected from light during experiment; store aliquoted at -20°C protected from light |
| NADPH Standard | Reconstitute with 1 mL deionized water before use to 10 mM concentration | Keep on ice protected from light during experiment; store aliquoted at -20°C protected from light |
| NADP Extraction Buffer | Ready-to-use; equilibrate to RT before use | Store at 4°C |
| NADPH Extraction Buffer | Ready-to-use; equilibrate to RT before use | Store at 4°C |
2. Standard Preparation
Pipette 2 µL of the 10 mM NADPH standard into 998 µL Assay Buffer to prepare 1 mL of 20 µM NADPH premix. Then dilute serially as shown in the table below:
| Standard Working Solution | Volume of 20 µM NADPH Premix (µL) | Volume of Assay Buffer (µL) | Final NADPH Concentration (µM) |
| 1 | 100 | 0 | 20 |
| 2 | 50 | 50 | 10 |
| 3 | 40 | 60 | 8 |
| 4 | 20 | 80 | 4 |
| 5 | 10 | 90 | 2 |
| 6 | 5 | 95 | 1 |
| 7 | 2.5 | 97.5 | 0.5 |
3. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. Thaw frozen samples on ice before use, noting that this may affect stability and result in lower than expected values. Avoid the presence of interfering substances such as: EDTA (>0.5 mM), ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%), and Tween-20 (>1%).
3.1 Tissue Samples
Rinse tissue with ice-cold PBS. Weigh about 20 mg of tissue, mince with scissors, and place in a homogenizer.
For NADP extraction: Add 100 µL NADP Extraction Buffer and homogenize. Boil for 5 min (tightly capped to prevent evaporation). Then add 20 µL Assay Buffer and 100 µL NADPH Extraction Buffer to neutralize the extract. Centrifuge at 4°C, 14,000 rpm for 5 min. Transfer the supernatant to a new tube and keep on ice for assay.
For NADPH extraction: Add 100 µL NADPH Extraction Buffer and homogenize. Boil for 5 min (tightly capped to prevent evaporation). Then add 20 µL Assay Buffer and 100 µL NADP Extraction Buffer to neutralize the extract. Centrifuge at 4°C, 14,000 rpm for 5 min. Transfer the supernatant to a new tube and keep on ice for assay.
3.2 Cell or Bacterial Samples
Collect 1×10⁶ cells/bacteria. Wash with cold PBS, centrifuge at 800 g for 2 min, and discard the supernatant.
For NADP extraction: Add 100 µL NADP Extraction Buffer. Sonicate for 5 min (20% power or 200 W, pulse 3s on, 7s off, repeat 30 times). Boil for 5 min (tightly capped). Then add 20 µL Assay Buffer and 100 µL NADPH Extraction Buffer to neutralize. Centrifuge at 4°C, 14,000 rpm for 5 min. Transfer the supernatant to a new tube and keep on ice for assay.
For NADPH extraction: Add 100 µL NADPH Extraction Buffer. Sonicate for 5 min (20% power or 200 W, pulse 3s on, 7s off, repeat 30 times). Boil for 5 min (tightly capped). Then add 20 µL Assay Buffer and 100 µL NADP Extraction Buffer to neutralize. Centrifuge at 4°C, 14,000 rpm for 5 min. Transfer the supernatant to a new tube and keep on ice for assay.
3.3 Serum/Plasma Samples
For NADP extraction: Pipette about 20 µL of serum/plasma. Add 100 µL NADP Extraction Buffer. Boil for 5 min (tightly capped). Then add 20 µL Assay Buffer and 100 µL NADPH Extraction Buffer to neutralize. Centrifuge at 4°C, 14,000 rpm for 5 min. Transfer the supernatant to a new tube and keep on ice for assay.
For NADPH extraction: Pipette about 20 µL of serum/plasma. Add 100 µL NADPH Extraction Buffer. Boil for 5 min (tightly capped). Then add 20 µL Assay Buffer and 100 µL NADP Extraction Buffer to neutralize. Centrifuge at 4°C, 14,000 rpm for 5 min. Transfer the supernatant to a new tube and keep on ice for assay.
4. Assay Procedure
4.1 Microplate Reader Preparation: Preheat for at least 30 minutes. Set the wavelength to 450 nm.
4.2 Working Solution Preparation: Prepare 85 µL working solution per well fresh. Mix 68 µL Assay Buffer, 1 µL NADP Cycling Enzyme Mix, 5 µL WST-8, and 1 µL Enhancer. Incubate at room temperature for 5 min. Then add 10 µL Glucose.
4.3 Assay System Setup: Pipette reagents into a 96-well plate as follows. This assay is based on an enzyme-catalyzed kinetic reaction; add working solution quickly and mix all components thoroughly. A multichannel pipette is recommended. Standard and Blank wells are typically set up in singlicate.
| Reagent | Blank Well (μL) | Standard Well (μL) | Test Well (μL) |
| Assay Buffer | 40 | 0 | 0 |
| Standard Working Solution | 0 | 40 | 0 |
| Sample | 0 | 0 | 40 |
| Working Solution | 80 | 80 | 80 |
4.4 Absorbance Measurement: Mix thoroughly. Measure the absorbance at 450 nm (A₁). Incubate at room temperature for 30 min. Measure the absorbance at 450 nm again (A₂).
5. Calculation of Results
The following derived formula and simplified formula are completely equivalent.
5.1 Data Processing
Calculate ΔA = A₂ - A₁.
Calculate ΔΔAstandard = ΔAstandard - ΔAblank
Calculate ΔΔAtest = ΔAtest - ΔAblank
5.2 Standard Curve Plotting
Plot the standard curve with the standard concentration (µM) on the Y-axis and ΔΔAstandard on the X-axis. Substitute ΔΔAtest into the equation to obtain the Y value (µM).
5.3 Sample NADP or NADPH Content Calculation
(1) Based on sample weight:
NADP or NADPH (nmol/g weight) = (Y × Vsample) ÷ (W × Vsample ÷ Vtotal) × n = 0.22 × Y ÷ W × n
(2) Based on cell/bacteria count:
NADP or NADPH (nmol/10⁴ cells) = (Y × Vsample) ÷ (N × Vsample ÷ Vtotal) × n = 0.22 × Y ÷ N × n
(3) Based on liquid volume:
NADP or NADPH (nmol/mL) = Y × (Vtotal + Vextract) ÷ Vextract × n = 12 × Y × n
(4) Based on protein concentration:
NADP or NADPH (nmol/mg prot) = (Y × Vsample) ÷ (Vsample × Cpr) × n = Y ÷ Cpr × n
Parameter Definitions:
nmol: 1 µM = 1 nmol/mL;
Vsample: Volume of sample added to well, 0.04 mL;
Vtotal: Total volume of tissue sample extract, 0.22 mL;
Vextract: Volume of serum/plasma used for extraction, 0.02 mL;
Cpr: Sample protein concentration, mg/mL;
W: Sample weight, g;
N: Cell/Bacteria number, in units of 10⁴ (e.g., if cell number is 1×10⁶, N=100);
n: Sample dilution factor.
6. Typical Results
Typical Standard Curve: Y=26.294X+0.0516, R²=0.9998
Note: The standard curves for NADP and NADPH are identical at these concentrations. This manual provides only the NADPH standard curve.
FAQ
Q: What should I do if the sample ΔAtest is too high or too low?
A: If the sample ΔΔAtest is higher than the ΔΔAstandard of the 20 µM standard, dilute the sample with deionized water before repeating the assay. If the sample ΔΔAtest is lower than the ΔΔAstandard of the 0.5 µM standard, increase the sample amount used for extraction.
Notes
1. It is recommended to perform a pilot experiment using 2-3 samples expected to have significant differences before formal testing.
2. For tissue and cell samples, results can be normalized by measuring protein concentration. For protein concentration determination, Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.
3. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.
4. It is recommended to establish a standard curve for each experiment to improve accuracy. If a standard curve is not run, refer to the typical standard curve formula in the 'Results' section for calculation.
5. Biochemical reagents are generally irritating and/or toxic. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, head cover, etc.) throughout the experiment and perform steps in a fume hood or biosafety cabinet when applicable.
6. This product is for research use only. Not for use in diagnostic procedures.
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