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.
Simple, direct and automation-ready procedures for measuring NAD+/NADH concentration are very desirable. NAD+/NADH assay kit is based on a lactate dehydrogenase cycling reaction, in which the formed NADH reduces a formazan (MTT) reagent. The intensity of the reduced product color, measured at 565 nm, is proportional to the NAD+/NADH concentration in the sample. This assay is highly specific for NAD+/NADH and with minimal interference (<1%) by NADP+/NADPH. Our assay 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 0.05uM and linearity up to 10uM NAD+/NADH in 96-well plate assay
Convenient. The procedure involves adding a single working reagent, and reading the optical density at time zero and 15 min at room temperature
High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.
Kit Contents:Assay Buffer: 10ml Enzyme A: 120ulLactate: 1.5ml Enzyme B: 120ulMTT Solution: 1.5ml NAD Standard: 0.5mlNAD/NADH Extraction Buffers: each 12mlStorage 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.
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.
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 Eppendorf tube with either 100ul NAD extraction buffer for NAD determination or 100ul NADH extraction buffer for NADH 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 NAD/NADH assays. Determination of both NAD and NADH concentrations requires extractions from two separate samples
2. Calibration Curve. Prepare 500ul 10uM NAD Premix by mixing 5ul 1mM Standard and 495ul distilled water. Dilute standard as follows
No Premix+H2O NAD (uM)1 100ul+0ul 102 60ul+40ul 63 30ul+70ul 34 0ul+100ul 0Transfer 40ul standards into wells of a clear flat-bottom 96-well plate
3. Samples. Add 40ul of each sample in separate wells
4. Reagent Preparation. For each well of reaction, prepare Working Reagent by mixing 60ul Assay Buffer, 1ul Enzyme A, 1ul Enzyme B, 14ul Lactate and 14ul MTT. Fresh reconstitution is recommended
5. Reaction. Add 80ul Working Reagent per well quickly. Tap plate to mix briefly and thoroughly
6. Read optical density (OD0) for time “zero” at 565 nm (520-600nm) and OD15 after a 15-min incubation at room temperature.
Calculation:First compute the DOD for each standard and sample by subtracting OD0 from OD15. Plot the standard DOD’s and determine the slope. The NAD(H) concentration of the sample is computed as follows:[NAD(H)] =DODSAMPLE–DODBLANKSlope (uM-1)× n (uM)where DODSAMPLE and DODBLANK are the change in optical density 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)
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.
