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

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.\n\nApplications:\nDirect Assays: NAD+/NADH concentrations and ratios in cell or tissue extracts.\n\nKey Features:\nSensitive and accurate. Detection limit of 0.02uM and linearity up to 1uM NAD+/NADH in 96-well plate assay.\nConvenient. The procedure involves adding a single working reagent, and reading the fluorescence at time zero and 10 min.\nHigh-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.\n\nKit Contents:\nAssay Buffer: 10ml Enzyme A: 120ul\nLactate: 1.5ml Enzyme B: 120ul\nProbe: 750ul NAD Standard: 0.5ml\nNAD/NADH 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\nGeneral Considerations:\n1. 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.\n2. 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.\n3. 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%).\n4. For samples containing higher than 100uM pyruvate, we recommend using an internal standard.\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 Eppendorf tube with either 100ul NAD extraction buffer for NAD determination or 100ul NADH extraction buffer for NADH 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 NAD/NADH assays. Determination of both NAD and NADH concentrations requires extractions from two separate samples.\n2. Calibration Curve. Prepare 500ul 1uM NAD Premix by mixing 5ul 1mM Standard and 4995ul distilled water. Dilute standard as follows.\nNo Premix+H2O NAD (uM)\n1 100ul+0ul 1.0\n2 60ul+40ul 0.6\n3 30ul+70ul 0.3\n4 0ul+100ul 0\nTransfer 50ul standards into wells of a clear flat-bottom 96-well plate.\n3. Samples. Add 50ul of each sample in separate wells.\n4. Reagent Preparation. For each reaction well, prepare Working Reagent by mixing 40ul Assay Buffer, 1ul Enzyme A, 1ul Enzyme B, 10ul Lactate and 5ul Probe. Fresh reconstitution is recommended.\n5. Reaction. Add 50ul Working Reagent per well quickly. Tap plate to mix.\n6. Read fluorescence at lex/em=530/585 nm for time пїЅzeroпїЅ (F0) and F10 after a 10-min incubation at room temperature. Protect plate from light during this incubation.\n\nCalculation:\nFirst compute the DF for each standard and sample by subtracting F0 from F10. Plot the standard DFпїЅs and determine the slope. The NAD(H) concentration of the sample is computed as follows:\n[NAD(H)] =\nDFSAMPLEпїЅDFBLANK\nSlope (uM-1)\nпїЅ n (uM)\nwhere DFSAMPLE and DFBLANK are the change in fluorescence intensity values of the Sample and Blank (STD 4) respectively. Slope is the slope of the standard curve and n is the dilution factor (if necessary).\nNote: If the sample DF values are higher than the DF value for the 1uM standard, dilute sample in distilled water and repeat this assay. Multiply the results by the dilution factor.\n\nMaterials Required, But Not Provided:\nPipetting (multi-channel) devices. Black, flat bottom 96-well plates and fluorescent plate reader capable of reading at lex/em=530/585 nm.

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SPECIFICATIONS

Catalog Number

N0009-19

Size

1Kit

References

1. Zhao, Z, Hu, X and Ross, CW (1987). Comparison of Tissue Preparation Methods for Assay of Nicotinamide Coenzymes. Plant Physiol. 84: 987-988.\n2. Matsumura, H. and Miyachi, S (1980). Cycling assay for nicotinamide adenine dinucleotides. Methods Enzymol. 69: 465-470.\n3. Vilcheze, C et al. (2005). Altered NADH/NAD+ Ratio Mediates Coresistance to Isoniazid and Ethionamide in Mycobacteria. Antimicrobial Agents and Chemotherapy. 49(2): 708-720.