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BioAssay Systems NAD+/NADH
EnzyFluoTM NAD+/NADH Assay Kit (EFND-100)
Quantitative Fluorimetric Determination of NAD+/NADH
Pyridine nucleotides play an important role in metabolism and, thus, there is
Premix + H2O
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
. 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.
. Add 50 µL Working Reagent per well quickly. Tap plate to mix.
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
Sensitive and accurate
. Detection limit of 0.02 µM and linearity up to 1 µM
First compute the ∆F for each standard and sample by subtracting F0 from
. 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:
. Can be readily automated as a high-throughput 96-well
plate assay for thousands of samples per day.
where ∆FSAMPLE and ∆FBLANK 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).
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
. 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.
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
[Pyridine Nucleotide] (
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 1.5 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.
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NORMAS CONDICIONALES Y FALACIA NATURALISTA* RESUMEN. Este trabajo muestra en qué medida la representación de las normas condicionales como «p → Oq» (la concepción puente de las normas condicionales) conduce a conclusiones paradó-jicas, por ejemplo, que un mundo proposicionalmente consistente es materialmente equivalentea mundos deónticamente contradictorios. Esta conclusión no