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BioAssay Systems NAD+/NADH EFND001.pdf
EnzyFluoTM NAD+/NADH Assay Kit (EFND-100)
Quantitative Fluorimetric Determination of NAD+/NADH
DESCRIPTION
Pyridine nucleotides play an important role in metabolism and, thus, there is Premix + H2O
NAD (µM)
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. BioAssay Systems' EnzyFluoTM 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 Transfer 50 µL standards into wells of a clear flat-bottom 96-well plate. λex/em = 530/585 nm, is proportional to the NAD+/NADH concentration in the sample. This assay is highly specific for NAD+/NADH with minimal 3. Samples. Add 50 µL of each sample in separate wells. interference (<1%) by NADP+/NADPH and is a convenient method to 4. Reagent Preparation. For each reaction well, prepare Working Reagent by mixing 40 µL Assay Buffer, 1 µL Enzyme A, 1 µL Enzyme B, 10 µL Lactate and 5 µL Probe. Fresh reconstitution is recommended. APPLICATIONS
5. Reaction. Add 50 µL Working Reagent per well quickly. Tap plate to mix. Direct Assays: NAD+/NADH concentrations and ratios in cell or tissue
ex/em = 530/585 nm for time “zero” (F0) and F10 after a 10-min incubation at room temperature. Protect plate from light KEY FEATURES
Sensitive and accurate. Detection limit of 0.02 µM and linearity up to 1 µM
CALCULATION
First compute the ∆F for each standard and sample by subtracting F0 from Convenient. The procedure involves adding a single working reagent, and
F10. Plot the standard ∆F’s and determine the slope. The NAD(H) reading the fluorescence at time zero and 10 min. concentration of the sample is computed as follows: High-throughput. Can be readily automated as a high-throughput 96-well
plate assay for thousands of samples per day.
KIT CONTENTS
Assay Buffer:
Enzyme A:
where ∆FSAMPLE and ∆FBLANK are the change in fluorescence intensity values Lactate:
Enzyme B:
of the Sample and Blank (STD 4) respectively. Slope is the slope of the standard curve and n is the dilution factor (if necessary). NAD Standard: 0.5 mL
NAD/NADH Extraction Buffers: each 12 mL
Note: If the sample ∆F values are higher than the ∆F value for the 1 µM standard, dilute sample in distilled water and repeat this assay. Multiply the Storage conditions. The kit is shipped on ice. Store all reagents at -20°C.
Shelf life: 6 months after receipt.
Precautions: reagents are for research use only. Normal precautions for
MATERIALS REQUIRED, BUT NOT PROVIDED
laboratory reagents should be exercised while using the reagents. Please Pipetting (multi-channel) devices. Black, flat bottom 96-well plates and refer to Material Safety Data Sheet for detailed information. fluorescent plate reader capable of reading at λex/em = 530/585 nm. 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 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 (>0.5 mM), ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%) and Tween-20 (>1%). 4. For samples containing higher than 100 µM pyruvate, we recommend PROCEDURES
[Pyridine Nucleotide] (µM)
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 LITERATURE
cells) in a 1.5 mL Eppendorf tube with either 100 µL NAD extraction 1. Zhao, Z, Hu, X and Ross, CW (1987). Comparison of Tissue Preparation buffer for NAD determination or 100 µL NADH extraction buffer for Methods for Assay of Nicotinamide Coenzymes. Plant Physiol. 84: 987- NADH determination. Heat extracts at 60°C for 5 min and then add 20 µL Assay Buffer and 100 µL of the opposite extraction buffer to 2. Matsumura, H. and Miyachi, S (1980). Cycling assay for nicotinamide neutralize the extracts. Briefly vortex and spin the samples down at adenine dinucleotides. Methods Enzymol. 69: 465-470. 14,000 rpm for 5 min. Use supernatant for NAD/NADH assays. 3. Vilcheze, C et al. (2005). Altered NADH/NAD+ Ratio Mediates Determination of both NAD and NADH concentrations requires Antimicrobial Agents and Chemotherapy. 49(2): 708-720. 2. Calibration Curve. Prepare 500 µL 1 µM NAD Premix by mixing 5 µL 1 mM Standard and 4995 µL distilled water. Dilute standard as follows. 2012  by BioAssay Systems · 3191 Corporate Place, Hayward, CA 94545, USA · Website: www.bioassaysys.com Tel: 510-782-9988, Fax: 510-782-1588 · Email: [email protected], [email protected]

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