Amplite® Fluorimetric Total NAD and NADH Assay Kit *Red Fluorescence*
|Shipping||Standard overnight for United States, inquire for international|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
Fluorescence microplate reader
|Recommended plate||Solid black|
AT A GLANCE
- Prepare NAD/NADH working solution (50 µL)
- Add NADH standards or test samples (50 µL)
- Incubate at room temperature for 15 minutes – 2 hours
- Monitor the fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)
Thaw one of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. NADH standard solution (1 mM):
Add 200 µL of 1X PBS buffer into the vial of NADH Standard (Component C) to make 1 mM (1 nmol/µL) NADH standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15257
Add 10 µL of 1 mM (1 nmol/µL) NADH standard solution to 990 µL 1X PBS buffer to generate 10 µM (10 pmol/µL) NADH standard solution (NS7). Take 10 µM NADH standard solution (NS7) to perform 1:3 serial dilutions in 1X PBS buffer to get serially diluted NADH standards (NS6 - NS1). Note: Diluted NADH standard solution is unstable and should be used within 4 hours.
PREPARATION OF WORKING SOLUTION
Add 10 mL of NADH Sensor Buffer (Component B) into the bottle of NAD/NADH Recycling Enzyme Mix (Component A) and mix well to make NAD/NADH working solution. Note: This NAD/NADH working solution is enough for two 96-well plates.
For guidelines on cell sample preparation, please visit
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of NADH standards and test samples in a solid black bottom 96-well microplate. NS=NADH Standards (NS1 - NS7, 0.014 to 10 µM) , BL=Blank Control, TS=Test Samples.
Table 2. Reagent composition for each well. High concentration of NADH (e.g., >100 µM, final concentration) may cause reduced fluorescence signal due to the over oxidation of NADH sensor (to a non-fluorescent product).
|NS1 - NS7||50 µL||Serial Dilutions (0.014 to 10 µM)|
|BL||50 µL||1X PBS buffer|
|TS||50 µL||test sample|
- Prepare NADH standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Note: Prepare cells or tissue samples as desired.
- Add 50 µL of NAD/NADH working solution to each well of NADH standard, blank control, and test samples to make the total NAD/NADH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of NAD/NADH working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at room temperature for 15 minutes to 2 hours, protected from light.
- Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm). Note:The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. The absorption detection has lower sensitivity compared to fluorescence reading. Note: For NAD/NADH ratio measurements, kit 15263 is recommended. Note: For cell based NAD/NADH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (cat#20012) is recommended to use for lysing the cells.
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