Pyridine nucleotides play an important role in metabolism and, thus, there is continual interest in monitoring their concentration levels. Quantitative determination of NADP+/NADPH has applications in research pertaining to energy transformation and redox state of cells or tissue.||Simple, direct and automation-ready procedures for measuring NADP+/NADPH concentration are very desirable. NADP+/NADPH assay kit is based on a glucose dehydrogenase cycling reaction, in which the formed NADPH reduces a formazan (MTT) reagent. The intensity of the reduced product color, 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 by NAD+/NADH. Our assay is a convenient method to measure NADP, NADPH and their ratio.||Applications:|Direct Assays: NADP+/NADPH concentrations and ratios in cell or tissue extracts.||Key Features:|Sensitive and accurate. Detection limit 0.1uM, linearity up to 10uM NADP+/NADPH in 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.||Kit Contents: (100 tests in 96-well plates)|Assay Buffer: 10ml Glucose (1 M): 1.5ml|MTT Solution: 1.5ml Enzyme Mix: 120ul|NADP Standard: 0.5ml 1mM|NADP/NADPH Extraction Buffers: each 12ml|Storage conditions. Store all reagents at -20°C. Shelf life: 6 months after receipt.|Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.||Procedures:|1. Sample Preparation. For tissues weigh ~20 mg tissue for each sample, wash with cold PBS. For cell samples, wash cells with cold PBS and pellet ~105 cells for each sample. Homogenize samples (either tissue or cells) in a 1.5ml eppindorf tube with either 100ul NADP extraction buffer for NADP determination or 100ul NADPH extraction buffer for NADPH determination. Heat extracts at 60°C for 5 min and then add 20ul Assay Buffer and 100ul of the opposite extraction buffer to neutralize the extracts. Briefly vortex and spin the samples down at 14,000 rpm for 5 min. Use supernatant for NADP/NADPH assays. Determination of both NADP and NADPH concentrations requires extractions from two separate samples.|2. Calibration Curve. Prepare 500ul 10uM NADP Premix by mixing 5ul 1mM Standard and 495ul distilled water.|No Premix+H2O Vol (ul) [NADP] (uM)|1 100ul+0ul 100 10|2 80ul+20ul 100 8|3 60ul+40ul 100 6|4 40ul+60ul 100 4|5 30ul+70ul 100 3|6 20ul+80ul 100 2|7 10ul+90ul 100 1|8 0ul+100ul 100 0|Dilute standard as shown in the Table. Transfer 40ul standards into wells of a clear bottom 96-well plate. Samples: add 40ul sample per well in separate wells.|3. Reagent Preparation. For best results allow Enzyme to come to RT (15-30 min) before preparing the Working Reagent. For each well of reaction, prepare Working Reagent by mixing 60ul Assay Buffer, 1ul Enzyme Mix, 10ul Glucose and 14ul MTT. Fresh reconstitution is recommended.|4. Reaction. Add 80ul Working Reagent per well quickly. Tap plate to mix briefly and thoroughly.|5. Read optical density (OD0) for time “zero” at 565 nm (520-600nm) and OD30 after a 30-min incubation at room temperature.|6. Calculation:. Subtract OD0 from OD30 for the standard and sample wells. Use the DOD values to determine sample NADP/NADPH concentration from the standard curve.|[NADP(H)] =|DODSAMPLE–DODBLANK|Slope (uM-1)|× n (uM)|Note: If the sample DOD values are higher than the DOD value for the 10uM standard, dilute sample in distilled water and repeat this assay. Multiply the results by the dilution factor.||Materials Required, But Not Provided:|Pipetting (multi-channel) devices. Clear-bottom 96-well plates (e.g. Corning Costar) and plate reader.||General Considerations:|1. At these concentrations, the standard curves for NADP and NADPH are identical. Since NADPH in solution is unstable, we provide only NADP as the standard.|2. This assay is based on an enzyme-catalyzed kinetic reaction. Addition of Working Reagent should be quick and mixing should be brief but thorough. Use of multi-channel pipettor is recommended.|3. The following substances interfere and should be avoided in sample preparation. EDTA (>0.5mM), ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%) and Tween-20 (>1%).