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Amplite® Fluorimetric Neuraminidase Assay Kit *Blue Fluorescence*
Example protocol
AT A GLANCE
Protocol summary
- Prepare Neuraminidase working solution (50 µL)
- Add Neuraminidase standards or test samples (50 µL)
- Incubate at 37°C or room temperature for 1 - 2 hours
- Monitor fluorescence increase at Ex/Em = 320/460 nm (Cutoff = 420 nm)
Important notes
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. Neuraminidase standard solution (2U/mL):
Add 50 µL of ddH2O into the vial of Neuraminidase Standard (Component C) to make approximately 2 U/mL Neuraminidase standard solution.
2. FluLite™ Blue stock solution (200X):
Add 50 µL of ddH2O into the vial of FluLite™ Blue (Component A) to make 200X stock solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/12602
Add 10 µL of 2 U/mL Neuraminidase standard stock solution to 990 µL of Assay Buffer (Component B) to generate 20 mU/mL Neuraminidase standard (NA7). Take 20 mU/mL Neuraminidase standard solution and perform 1:2 serial dilutions to get serially diluted Neuraminidase standards (NA6 - NA1) with Assay Buffer (Component B). Note: Diluted Neuraminidase standard solution is unstable. Use within 4 hours.
PREPARATION OF WORKING SOLUTION
Add 25 μL of 200X FluLite™ Blue stock solution into 5 mL of Assay Buffer (Component B) and mix well to prepare Neuraminidase working solution.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of neuraminidase standards and test samples in a solid black 96-well microplate. NA= Neuraminidase Standards (NA1 - NA7, 0.312 to 20 mU/mL), BL=Blank Control, TS=Test Samples.
| BL | BL | TS | TS |
| NA1 | NA1 | ... | ... |
| NA2 | NA2 | ... | ... |
| NA3 | NA3 | ||
| NA4 | NA4 | ||
| NA5 | NA5 | ||
| NA6 | NA6 | ||
| NA7 | NA7 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| NA1 - NA7 | 50 µL | Serial Dilution (0.312 to 20 mU/mL) |
| BL | 50 µL | Blank Control |
| TS | 50 µL | test sample |
- Prepare Neuraminidase standards (NA), 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.
- Add 50 µL of Neuraminidase working solution to each well of Neuraminidase standard, blank control, and test samples to make the total Neuraminidase assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Neuraminidase working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at 37°C or room temperature for 1 to 2 hours, protected from light. Note: 37°C incubation gives better results.
- Monitor the fluorescence increase at Ex/Em = 320/460 nm (Cutoff = 420 nm) with a fluorescence microplate reader.
Spectrum
References
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Journal: J Sep Sci (2016): 2097
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Journal: Vaccine (2016): 3773
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Journal: J Mol Recognit (2015): 87
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Journal: Zhonghua Yu Fang Yi Xue Za Zhi (2015): 481
Authors: Pedersen JC., undefined
Journal: Methods Mol Biol (2014): 27
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