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Amplite® Fluorimetric Mercuric Ion Quantitation Kit
Example protocol
AT A GLANCE
Protocol summary
- Prepare mercury working solution (50 µL)
- Add mercury (II) standard or test samples (50 µL)
- Incubate at room temperature for 20 - 30 minutes
- Monitor fluorescence intensity at Ex/Em = 540/590 nm
Important notes
To achieve the best results, it’s strongly recommended to use the black plates. Thaw kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. Mercury Lite™ 590 stock solution (200X):
Add 25 µL of DMSO into the vial of Mercury Lite™ 590 (Component A) and mix them well. Keep from light. Note: Make single use aliquots, and store unused 200X Mercury Lite™ 590 stock solution at -20ºC, avoid light and repeat freeze-thaw cycles.
2. Mercury (II) standard stock solution (not provided):
We used Mercury (II) Perchlorate hydrate (Sigma 529656, CAS#304656-34-6) as the mercury (II) standard. The stock solution of mercury (II) was prepared at the concentration of 1 mM in ddH2O.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/19005
Perform 1:2 serial dilutions using ddH2O to get approximately 500, 250, 125, 62.5, 31.3, 15.6 and 7.8 µM serially diluted mercury (II) standards (M7 - M1).
PREPARATION OF WORKING SOLUTION
Add 25 µL of Mercury Lite™ 590 stock solution into 5 mL of Assay Buffer (Component B) and mix them well (Component A+B). Note: This mercury working solution is enough for one 96-well plate. It is not stable, use it promptly.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of mercury (II) standards and test samples in a solid black 96-well microplate. M= Mercury (II) Standards (M1 - M7, 7.8 to 500 µM), BL=Blank Control, TS=Test Samples.
| BL | BL | TS | TS |
| M1 | M1 | ... | ... |
| M2 | M2 | ... | ... |
| M3 | M3 | ||
| M4 | M4 | ||
| M5 | M5 | ||
| M6 | M6 | ||
| M7 | M6 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| M1 - M7 | 50 µL | Serial Dilutions (7.8 to 500 µM) |
| BL | 50 µL | Assay Buffer |
| TS | 50 µL | test sample |
- Prepare and add mercury (II) standards (M), 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 mercury working solution to each well of mercury (II) standard, blank control, and test samples to make the total mercury assay volume of 100 µL/well. For a 384-well plate, add 25 µL of mercury working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at room temperature for 20 - 30 minutes, protected from light.
- Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm, cutoff 570 nm.
Product family
| Name | Excitation (nm) | Emission (nm) |
| Amplite® Fluorimetric Zinc Ion Quantitation Kit | 493 | 516 |
References
Authors: Chen, Xiaojun and Zu, Yanbing and Xie, Hong and Kemas, Aurino Muhammad and Gao, Zhiqiang
Journal: Analyst (2011): 1690--1696
Authors: Hong, Baoyu and Nauss, Rachel and Harwood, Ian M and Miller, Susan M
Journal: Biochemistry (2010): 8187--8196
Authors: Schué, Mathieu and Dover, Lynn G and Besra, Gurdyal S and Parkhill, Julian and Brown, Nigel L
Journal: Journal of bacteriology (2009): 439--444
Authors: Rossy, Emmanuel and Sénèque, Olivier and Lascoux, David and Lemaire, David and Crouzy, Serge and Delangle, Pascale and Covès, Jacques
Journal: FEBS letters (2004): 86--90
Authors: Wilson, Jon R and Leang, Ching and Morby, Andrew P and Hobman, Jon L and Brown, Nigel L
Journal: FEBS letters (2000): 78--82
Safety data sheet