Description
NADP/NADPH Assay Kit (BA0232) (BA0232)
Pyridine nucleotides play an important role in metabolism, and there is continual interest in monitoring their concentration levels. Quantitative determination of NADP+/NADPH has applications in research pertaining to energy transformation and the redox state of cells or tissue. The NADP/NADPH Assay Kit (SKU: BA0232) is based on a glucose dehydrogenase cycling reaction, in which the formed NADPH reduces a formazan (MTT) reagent. The intensity of the reduced product colour, measured at 565 nm, is proportionate to the NADP+/NADPH concentration in the sample. This assay is highly specific for NADP+/NADPH and is not interfered with by NAD+/NADH, providing a convenient method to measure NADP, NADPH and their ratio. The single-reagent, room-temperature procedure is read at time zero and 30 minutes and is readily automated for high-throughput analysis.
| Product Name: | NADP/NADPH Assay Kit (BA0232) |
| SKU: | BA0232 |
| Detection Method: | Colorimetric determination at 565 nm (520-600 nm) |
| Detection Range: | Detection limit 0.1 µM, linearity up to 10 µM NADP+/NADPH in a 96-well plate assay |
| Sample Type: | Cell or tissue extracts |
| Species Reactivity: | All |
| Assay Time: | 30 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | The kit is shipped on ice. Store all reagents at -20°C. |
| Shelf Life: | 6 months after receipt. |
| Shipping: | Gel Pack |
An ultrasensitive colorimetric assay for NADP+/NADPH based on a glucose dehydrogenase cycling reaction in which the formed NADPH reduces a formazan (MTT) reagent. The intensity of the reduced product colour at 565 nm is proportional to the NADP+/NADPH concentration in the sample and is not interfered with by NAD+/NADH.
- Sensitive and accurate. Detection limit 0.1 µM, linearity up to 10 µM NADP+/NADPH in a 96-well plate assay.
- Convenient. The procedure involves adding a single working reagent and reading the optical density at time zero and 30 min at room temperature. No 37°C heater is required.
- High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.
- Direct assays: NADP+/NADPH concentrations and ratios in cell or tissue extracts.
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 per sample and wash with cold PBS; for cells wash with cold PBS and pellet ~10⁵ cells. Homogenise in a 1.5 mL tube with 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. Vortex, spin down at 14,000 rpm for 5 min and use supernatant. Determination of both NADP and NADPH requires extractions from two separate samples. |
| 2 | Calibration curve: prepare 500 µL 10 µM NADP Premix by mixing 5 µL 1 mM Standard and 495 µL distilled water. Dilute standards as per the table and transfer 40 µL standards into wells of a clear-bottom 96-well plate. |
| 3 | Samples: add 40 µL sample per well in separate wells. |
| 4 | Reagent preparation: allow Enzyme to reach room temperature (15-30 min). For each reaction well prepare Working Reagent by mixing 80 µL Assay Buffer, 1 µL Enzyme A, 1 µL Enzyme B, 1 µL G6P and 14 µL MTT. Fresh reconstitution is recommended. |
| 5 | Reaction: add 80 µL Working Reagent per well quickly. Tap plate to mix briefly and thoroughly. |
| 6 | Read optical density (OD0) at time zero at 565 nm (520-600 nm) and OD30 after a 30-min incubation at room temperature. |
Subtract OD0 from OD30 for the standard and sample wells. Use the ∆OD values to determine sample concentration from the standard curve: [NADP(H)] = [(∆ODSample – ∆ODBlank) / Slope (µM⁻¹)] × n (µM). If sample ∆OD values are higher than that of the 10 µM standard, dilute sample in distilled water and repeat, multiplying by the dilution factor.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20°C |
| Enzyme A | 120 µL | -20°C |
| MTT Solution | 1.5 mL | -20°C |
| Enzyme B | 120 µL | -20°C |
| G6P | 120 µL | -20°C |
| NADP Standard (1 mM) | 0.5 mL | -20°C |
| NAD(P)/NAD(P)H Extraction Buffers | each 12 mL | -20°C |
