BioChain NAD+/NADH V002 NAD+/NADH Assay Kit (Z5030037) Ultrasens itive Colorimetric Determination of NAD+/NADH at 565 nm DESCRIPTION
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
Samples. 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
4. Reaction. 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.
6. Calculation. 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.
Direct Assays: NAD+/NADH concentrations and ratios in cel or tissue
Multiply the results by the dilution factor.
MATERI ALS REQUIRED, BUT NOT PROVIDED KEY FEATURES
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 Convenient. The procedure involves adding a single w orking reagent, GENERAL CONSIDERATIONS
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
High-throughput. 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 LEnzyme 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%).
Storage conditions. Store al 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 w hile using the reagents.
Please refer to Material Safety Data Sheet for detailed information.
PROCEDURES 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.
Standard Curve 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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