NAD+/NADH Assay Kit (Z5030037)
Ultrasens itive Colorimetric Determination of NAD+/NADH at 565 nm
Transfer 40 mL standards into w el s of a clear flat-bottom 96-w el
Pyr idine nuc leotides play an important role in metabolis m and,
thus, ther e is continual interest in monitoring their concentration
. Add 40 mL sample per w el in separate w el s.
levels. Quantitative determination of NAD+/NADH has applications in
3. Reagent Preparation
. For each w el of reaction, prepare Working
research pertaining to energy transformation and redox state of cel s
Reagent by mixing 60 mL Assay Buffer, 1 mL Enzyme A, 1 mL
Enzyme B, 14 mL Lactate and 14 mL MTT. Fresh reconstitution is
Simple, direct and automation-ready procedures for measuring
. Add 80 mL Working Reagent per w el quickly. Tap plate
NAD+/NADH assay kit is based on a lactate dehydrogenase cycling
reaction, in w hich the formed NADH reduces a formazan (MTT)
5. Read optical density (OD0) for time “zero” at 565 nm (520-600nm)
reagent. The intensity of the reduced product color, measured at 565
and OD15 after a 15-min incubation at room temperature.
nm, is proportionate to the NAD+/NADH concentration in the sample.
Subtract OD0 from OD15 for the standard and sample
This assay is highly specific for NAD+/NADH and w ith minimal
w el s. Use the DOD values to determine sample NAD/NADH
interference (<1%) by NADP+/NADPH. Our assay is a convenient
method to measure NAD, NADH and their ratio.
Note: If the sample DOD values are higher than the DOD value for the
10 mM standard, dilute sample in distil ed w ater and repeat this assay.
NAD+/NADH concentrations and ratios in cel or tissue
Multiply the results by the dilution factor.
MATERI ALS REQUIRED, BUT NOT PROVIDED
Pipetting (multi-channel) devices. Clear-bottom 96-w el plates (e.g.
Sensitive and accurate
. Detection limit 0.05 mM, linearity up to 10 mM
. The procedure involves adding a single w orking reagent,
and reading the optical density at time zero and 15 min at room
1. At these concentrations, the standard curves for NAD and NADH
temperature. No 37°C heater is required.
are identical. Since NADH in solution is unstable, w e provide only
. Can be readily automated as a high-throughput 96-
w el plate assay for thousands of samples per day.
2. This assay is based on an enzyme-catalyzed kinetic reaction.
KIT CONTENTS (100 te sts in 96-well plates)
Addition of Working Reagent should be quick and mixing should be
Assay Buffer: 10 mL Lactate: 1.5 mL
brief but thorough. Use of multi-channel pipettor is recommended.
MTT Solution: 1.5 m L Enzyme A: 120
3. The fol ow ing substances interfere and should be avoided in
sample preparation. EDTA (>0.5 mM), ascorbic acid, SDS (>0.2%),
NAD Standard: 0.5 m L 1 mM Enzyme B: 120 mL
NAD/NADH Extraction Buffers: each 12 mL
sodium azide, NP-40 (>1%) and Tw een-20 (>1%).
. Store al reagents at -20°C. Shelf life: 6 months
: reagents are for research use only. Normal precautions
for laboratory reagents should be exercised w hile using the reagents.
Please refer to Material Safety Data Sheet for detailed information.
1. Sample Preparation
. For tissues w eigh ~20 mg tissue for each
sample, w ash w ith cold PBS. For cel samples, w ash cel s w ith cold
PBS and pellet ~105 cel s for each sample. Homogenize samples
(either tissue or cel s) in a 1.5 mL Eppindorf tube w ith either 100 mL NAD extraction buffer for NAD determination or 100 mL NADH
extraction buffer for NADH determination. Heat extracts at 60°C for
5 min and then add 20 mL Assay Buffer and 100 mL of the opposite extraction buffer to neutralize the extracts. Briefly vortex and spin
[Pyridine Nucle otide ] (mM )
the samples dow n at 14,000 rpm for 5 min. Use supernatant for NAD/NADH assays. Determination of both NAD and NADH
concentrations requires extractions from tw o separate samples.
in 96-w el plate assay
2. Calibration Curve
. Prepare 500 mL 10 mM NAD Premix by mixing 5
mL 1 mM Standard and 495 mL distilled w ater. Dilute standard as
1. Zhao, Z, Hu, X and Ross CW (1987). Comparison of Tissue
Preparation Methods for Assay of Nicotinamide Coenzymes. Plant
2. Matsumura, H. and Miyachi S (1980). Cycling assay for
nicotinamide adenine dinucleotides. Methods Enzymol. 69: 465-470.
3. 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.
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