NADP/NADPH Assay Kit (Nicotinamide Adenine Dinucleotide Phosphate), BioAssay(TM)

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.\n\nSimple, 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.\n\nApplications:\nDirect Assays: NADP+/NADPH concentrations and ratios in cell or tissue extracts.\n\nKey Features:\nSensitive and accurate. Detection limit 0.1uM, linearity up to 10uM NADP+/NADPH in 96-well plate assay.\nConvenient. The procedure involves adding a single working reagent, and reading the optical density at time zero and 30 min at room temperature. No 37 degrees C heater is required.\nHigh-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.\n\nKit Contents: (100 tests in 96-well plates)\nAssay Buffer: 10ml Glucose (1 M): 1.5ml\nMTT Solution: 1.5ml Enzyme Mix: 120ul\nNADP Standard: 0.5ml 1mM\nNADP/NADPH Extraction Buffers: each 12ml\nStorage conditions. Store all reagents at -20 degrees C. Shelf life: 6 months after receipt.\nPrecautions: 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.\n\nProcedures:\n1. 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 degrees 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.\n2. Calibration Curve. Prepare 500ul 10uM NADP Premix by mixing 5ul 1mM Standard and 495ul distilled water.\nNo Premix+H2O Vol (ul) [NADP] (uM)\n1 100ul+0ul 100 10\n2 80ul+20ul 100 8\n3 60ul+40ul 100 6\n4 40ul+60ul 100 4\n5 30ul+70ul 100 3\n6 20ul+80ul 100 2\n7 10ul+90ul 100 1\n8 0ul+100ul 100 0\nDilute 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.\n3. 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.\n4. Reaction. Add 80ul Working Reagent per well quickly. Tap plate to mix briefly and thoroughly.\n5. Read optical density (OD0) for time

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SPECIFICATIONS

Catalog Number

N0009-73

Size

1Kit

References

1. Ding X et al (2009). Enhanced HtrA2/Omi expression in oxidative injury to retinal pigment epithelial cells and murine models of neurodegeneration. Invest Ophthalmol Vis Sci. 50(10): 4957-66.\n2. Tseng HC et al (2009). Metabolic engineering of Escherichia coli for enhanced production of (R)- and (S)-3-hydroxybutyrate. Appl Environ Microbiol. 75(10): 3137-45.\n3. Du J et al (2010). Mechanisms of ascorbate-induced cytotoxicity in pancreatic cancer. Clin Cancer Res. 16(2): 509-20.