Amplite® Fluorimetric 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 NADH working solution (50 µL)
- Add NADH standards or test samples (50 µL)
- Incubate at room temperature for 15 minutes - 2 hours
- Monitor fluorescence intensity at Ex/Em = 540/590 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 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/15261
Take the NADH standard solution and use PBS buffer to generate 100 µM NADH standard solution (NS7). Then perform 1:3 serial dilutions to get remaining serial dilutions of NADH standard (NS6 - NS1). Note: Diluted NADH standard solution is unstable, and should be used within 4 hours.
PREPARATION OF WORKING SOLUTION
Add 10 mL of Amplite™ NADH Assay Buffer (Component B) to the bottle of NADH Recycling Enzyme Mixture (Component A) and mix well. Note: This NADH working solution is enough for two 96-well or four 384-well plates. The working solution is not stable, use it promptly and avoid direct exposure to light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of NADH standards and test samples in a solid black 96-well microplate. NS = NADH standard (NS1 - NS7, 0.1 to 100 µM); BL = blank control; TS = test sample.
Table 2. Reagent composition for each well.
|NS1 - NS7||50 µL||Serial Dilution (0.1 to 100 µM)|
|TS||50 µL||Test Sample|
- Prepare NADH standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
- Add 50 µL of NADH working solution into each well of NADH standard, blank control, and test samples to make the total NADH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of 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 Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal 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. However, the absorption detection will have a lower sensitivity compared to that of the fluorescence reading. For cell based NADH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (Cat No. 20012) is recommended to use for lysing the cells.
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