NAD/NADH Assay Kit

Catalog No. ABIN1019680

Product Details

Target: NAD/NADH

Specificity: 0.02 μM

Characteristics: Sensitive and accurate. Detection limit of 0.02 µM and linearity up to 1 µM NAD + /NADH in 96-well plate assay. Convenient. The procedure involves adding a single working reagent, and reading the fluorescence at time zero and 10 min. High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day

Components: Assay Buffer: 10 mL. Enzyme A: 120 µL. Lactate: 1.5 mL. Enzyme B: 120 µL. Probe: 750 µL. NAD Standard: 0.5 mL. NAD/NADH Extraction Buffers: each 12 m

Material not included: Pipetting (multi-channel) devices. Black, flat bottom 96-well plates and fluorescent plate reader capable of reading at ex/em = 530/585 nm.

Application Details

Application Notes: Direct Assays: NAD + /NADH concentrations and ratios in cell or tissue extracts

Comment:

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 (>0.5 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 using an internal standard

Protocol: Prepare 500 µL 1 µM NAD Premix by mixing 5 µL 1 mM Standard and 4995 µL distilled water.
Samples: Add 50 µL of each sample in separate wells.
Reaction: Add 50 µL Working Reagent per well quickly. Tap plate to mix. 6. Read fluorescence at ex/em = 530/585 nm for time zero (F0) and F10 after a 10-min incubation at room temperature. Protect plate from light during this incubation

Reagent Preparation:

For each reaction well, prepare Working Reagent by mixing 40 µL Assay Buffer, 1 µL Enzyme A, 1 µL Enzyme B, 10x L Lactate and 5 µL Probe. Fresh reconstitution is recommended.

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 ~10 5 cells for each sample. Homogenize samples (either tissue or cells) in a 1.5 mL Eppendorf tube with either 100 µL NAD extraction buffer for NAD determination or 100 µL NADH extraction buffer for 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 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.

Calculation of Results:

First compute the F for each standard and sample by subtracting F0 from F10. Plot the standard F's and determine the slope. The NAD(H) concentration of the sample is computed as follows:
[NAD(H)] = [(F_SAMPLE - F_BLANK) / (Slope (µM-1))] x n (µM)
where F_SAMPLE and F_BLANK 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).
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 results by the dilution factor

Restrictions: For Research Use only

Handling

Storage: -20 °C

Target Details

Target: NAD/NADH

Target Type: Chemical

Background: Sensitive determination of NAD and NADH by fluorometric (530/585nm) method. Procedure: 10 min.

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