NAD/NADH Assay Kit (Nicotinamide Adenine Dinucleotide), BioAssay™, Colorimetric/Fluorometric

Pyridine nucleotides play an important role in metabolism and, thus, there is continual interest in monitoring their concentration levels. Quantitative determination of NAD+/NADH has applications in research pertaining to energy transformation and redox state of cells or tissue. NAD+/NADH assay kit is based on a lactate dehydrogenase cycling reaction, in which the formed NADH reduces a probe into a highly fluorescent product. The fluorescence intensity of this product, measured at lex/em=530/585 nm, is proportional to the NAD+/NADH concentration in the sample. This assay is highly specific for NAD+/NADH with minimal interference (<1%) by NADP+/NADPH and is a convenient method to measure NAD, NADH and their ratio.||Applications:|Direct Assays: NAD+/NADH concentrations and ratios in cell or tissue extracts.||Key Features:|Sensitive and Accurate: Detection limit of 0.02uM and linearity up to 1uM NAD+/NADH in 96-well plate assay.|Convenient: The procedure involves adding a single working reagent, and reading the fluorescence at time zero and 10 min.|High-throughput: Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.||Kit Components:|N0009-19A: Assay Buffer, 1x10ml |N0009-19B: Lactate, 1x1.5ml|N0009-19C: Probe, 1x750ul|N0009-19D: Enzyme A, 1x120ul|N0009-19E: Enzyme B, 1x120ul|N0009-19F: NAD Standard, 1x500ul|N0009-19G: NAD Extraction Buffer, 1x12ml|N0009-19H: NADH Extraction Buffer, 1x12ml||Storage and Stability:|Store all reagents at -20°C. Avoid freeze/thaw cycles. Stable for 6 months after receipt.||Precautions:|Reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents.||General Considerations:|1. At these concentrations, the standard curves for NAD and NADH are identical. Since NADH in solution is unstable, we provide only NAD 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%).|4. For samples containing higher than 100uM pyruvate, we recommend using an internal standard.