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, 1x1.5mlN0009-19C: Probe, 1x750ulN0009-19D: Enzyme A, 1x120ulN0009-19E: Enzyme B, 1x120ulN0009-19F: NAD Standard, 1x500ulN0009-19G: NAD Extraction Buffer, 1x12mlN0009-19H: 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.
