Amplite® Fluorimetric NAD Assay Kit *Blue 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 standards or test samples (50 µL)
- Add 20 µL Quest Fluor™ NAD Probe
- Add 20 µL Assay Solution
- Incubate at RT for 10 - 20 minutes
- Add 15 µL Enhancer Solution
- Incubate at RT for 10 - 20 min
- Monitor Fluorescence at 420/480 nm
Thaw each kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. NAD standard solution (1 mM):
Add 500 µL of ddH2O into the vial of NAD standard (Component D) to make 1 mM NAD stock solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15280
Add 10 µL of NAD standard solution into 990 µL H2O or PBS buffer to generate 10 µM NAD standard solution (NS7). Then take the 10 µM NAD standard solution and perform 1:3 serial dilutions in H2O or PBS to get remaining serial dilutions of NAD standard (NS1 - NS6). Note: Diluted NAD standard solution is unstable, and should be used within 4 hours.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of NAD standards and test samples in a black/solid bottom 96-well microplate. NS = NAD standard (NS1-NS7, 0.01 to 10 µM); BL = blank control; TS = test sample.
Table 2. Reagent composition for each well.
|NS1-NS7||50 µL||serial dilution (0.01 to 10 µM)|
|BL||50 µL||Assay Buffer|
- Prepare NAD standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Table 1 and Table 2. For 384-well plate, use 25 µL of reagent per well instead of 50 µL.
- Add 20 µL Quest Fluor™ NAD Probe (Component A) solution into each well of NAD standard, blank control, and test samples, mix well. For 384-well plate, use 10 µL of Quest Fluor™ NAD Probe (Component A) solution instead.
- Add 20 µL Assay Solution (Component B) into each well, mix well. For 384-well plate, use 10 µL of Assay Solution (Component B) instead.
- Incubate the reaction at room temperature for 10 - 20 minutes, protected from light.
- Add 15 µL Enhancer (Component C) to each well to make the total NAD assay volume of 105 µL/well, and incubate at room temperature for 10 - 20 minutes, protected from light. For a 384-well plate, add 7.5 uL Enhancer (Component C) instead, for a total volume of 52.5 µL/well.
- Monitor the fluorescence increase with a fluorescence plate reader at 420/480 nm.
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