Description
NADP/NADPH Assay Kit (BA0238) (BA0238)
The NADP/NADPH Assay Kit (SKU: BA0238) provides a simple, sensitive and automation-ready fluorimetric method for the quantitative determination of NADP+ and NADPH in biological samples. Pyridine nucleotides play an important role in cellular metabolism, and their quantitative measurement has wide applications in research into energy transformation and the redox state of cells and tissue. The assay is based on a glucose dehydrogenase cycling reaction, in which the formed NADPH reduces a probe into a highly fluorescent product measured at λex/em = 530/585 nm, the intensity of which is proportional to the NADP+/NADPH concentration in the sample. The assay is highly specific for NADP+/NADPH with minimal interference (<1%) from NAD+/NADH. It offers a convenient means of measuring NADP+, NADPH and their ratio for thousands of samples per day.
| Product Name: | NADP/NADPH Assay Kit (BA0238) |
| SKU: | BA0238 |
| Detection Method: | Fluorimetric (FL530/585 nm) |
| Detection Range: | 0.01 µM to 1 µM NADP+/NADPH (96-well plate) |
| Sample Type: | ['Cell extracts', 'Tissue extracts'] |
| Species Reactivity: | All |
| Assay Time: | 30 min |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20°C |
| Shelf Life: | 12 months |
| Shipping: | Gel Pack |
A fluorimetric assay kit for the quantitative determination of NADP+, NADPH and their ratio in cell and tissue extracts. The kit uses a glucose dehydrogenase cycling reaction that generates a highly fluorescent product measured at λex/em = 530/585 nm, with a detection limit of 0.01 µM and linearity up to 1 µM in a 96-well plate format.
- Sensitive and accurate. Detection limit of 0.01 µM and linearity up to 1 µM NADP+/NADPH in a 96-well plate assay.
- Convenient. The procedure involves adding a single working reagent and reading fluorescence at time zero and 30 min. Room temperature assay.
- High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.
- Direct assays of NADP+/NADPH concentrations and ratios in cell or tissue extracts.
- Research into energy transformation and the redox state of cells and tissue.
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Sample Preparation. For tissues, weigh ~20 mg tissue per sample and wash with cold PBS. For cells, wash with cold PBS and pellet ~10^5 cells per sample. Homogenise samples in a 1.5 mL tube with either 100 µL NADP+ extraction buffer (for NADP+) or 100 µL NADPH extraction buffer (for NADPH). Heat extracts at 60°C for 5 min, then add 20 µL Assay Buffer and 100 µL of the opposite extraction buffer to neutralise. Briefly vortex and spin down at 14,000 rpm for 5 min; use the supernatant for the assay. Determination of both NADP+ and NADPH requires extractions from two separate samples. |
| 2 | Calibration Curve. Prepare 5000 µL of 1 µM NADP+ Premix by mixing 5 µL of 1 mM Standard with 4995 µL distilled water. Dilute the standard to give 1.0, 0.6, 0.3 and 0 µM points (see Standard Dilution table). Transfer 50 µL standards into wells of a black flat-bottom 96-well plate. |
| 3 | Samples. Add 50 µL sample per well in separate wells. |
| 4 | Reagent Preparation. For each reaction well, prepare Working Reagent by mixing 45 µL Assay Buffer, 1 µL Enzyme A, 1 µL Enzyme B, 1 µL G6P and 5 µL Probe. Fresh reconstitution is recommended. |
| 5 | Reaction. Add 50 µL Working Reagent per well quickly. Tap plate to mix. |
| 6 | Read fluorescence at λex/em = 530/585 nm for time zero (F0) and again (F30) after a 30-min incubation at room temperature. Protect the plate from light during incubation. |
Compute the ΔF for each standard and sample by subtracting F0 from F30. Plot the standard ΔF values and determine the slope. [NADP(H)] = (ΔF_SAMPLE – ΔF_BLANK) / Slope (µM^-1) × n (µM), where ΔF_SAMPLE and ΔF_BLANK are the changes in fluorescence intensity of the Sample and Blank (STD 4) respectively, Slope is the slope of the standard curve and n is the dilution factor. If sample ΔF values exceed that of the 1 µM standard, dilute the sample in distilled water and repeat, multiplying the result by the dilution factor.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20°C |
| Enzyme A | 120 µL | -20°C |
| G6P | 120 µL | -20°C |
| Enzyme B | 120 µL | -20°C |
| Probe | 750 µL | -20°C |
| NADP Standard | 0.5 mL | -20°C |
| NADP Extraction Buffer | 12 mL | -20°C |
| NADPH Extraction Buffer | 12 mL | -20°C |
