Amplite® Fluorimetric NADPH 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|
|Component A: NADPH Recycling Enzyme Mixture||2 bottles (lyophilized powder)|
|Component B: NADPH Assay Buffer||1 bottle (20 mL)|
|Component C: NADPH Standard||1 vial (167 µg)|
AT A GLANCE
- Prepare NADPH working solution (50 µL)
- Add NADPH standards or test samples (50 µL)
- Incubate at room temperature for 15 minutes - 2 hours
- Monitor fluorescence increase at Ex/Em = 540/590 nm
Thaw one of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. NADPH standard solution (1 mM):
Add 200 µL of PBS buffer into the vial of NADPH standard (Component C) to make 1 mM (1 nmol/µL) NADPH stock solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15262
Use NADPH standard solution and PBS buffer (pH 7.4) to generate 100 µM (100 pmol/µL) NADPH standard solution (NS7). Then use 100 µM NADPH standard solution to perform 1:3 serial dilutions to get remaining serial dilutions of NADPH standard (NS6 - NS1). Note: Diluted NADPH standard solution is unstable, and should be used within 4 hours.
PREPARATION OF WORKING SOLUTION
Add 10 mL of Amplite™ NADPH Assay Buffer (Component B) into the bottle of NADPH Recycling Enzyme Mixture (Component A); mix well. Note: This NADPH working solution is enough for two 96-well plates. The working solution is not stable, use it promptly and avoid direct exposure to light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of NADPH standards and test samples in a solid black 96-well microplate. NS = NADPH 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 NADPH standards (NS), blank controls (BL), and test samples (TS) into a solid black 96-well microplate 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. Prepare cells or tissue samples as desired.
- Add 50 µL of NADPH working solution to each well of NADPH standard, blank control, and test samples to make the total NADPH assay volume of 100 µL/well. For a 384-well plate, use 25 µL of 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 Ex/Em = 540/590 nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by absorbance microplate reader at the wavelength of 576 ± 5 nm. However, the absorption detection will have a lower sensitivity compared to fluorescence reading. For cell based NADPH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (Cat No. 20012) is recommended to use for lysing the cells.
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