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Amplite® Fluorimetric Mercuric Ion Quantitation Kit

Hg2+ was measured with the Amplite® Fluorimetric Mercuric Ion Quantitation Kit (Cat#19005) in a 96-well solid black plate using a Gemini microplate reader (Molecular Devices). As low as 8 µM mercury (II) perchlorate was detected with 30 minutes incubation.
Hg2+ was measured with the Amplite® Fluorimetric Mercuric Ion Quantitation Kit (Cat#19005) in a 96-well solid black plate using a Gemini microplate reader (Molecular Devices). As low as 8 µM mercury (II) perchlorate was detected with 30 minutes incubation.
Hg2+ was measured with the Amplite® Fluorimetric Mercuric Ion Quantitation Kit (Cat#19005) in a 96-well solid black plate using a Gemini microplate reader (Molecular Devices). As low as 8 µM mercury (II) perchlorate was detected with 30 minutes incubation.
Hg2+ was measured with the Amplite® Fluorimetric Mercuric Ion Quantitation Kit (Cat#19005) in a 96-well solid black plate using a Gemini microplate reader (Molecular Devices). As low as 1.6 ppm mercury (II) perchlorate was detected with 30 minutes incubation.
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Telephone1-800-990-8053
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H-phraseH303, H313, H340
Hazard symbolT
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R68
UNSPSC41116134

OverviewpdfSDSpdfProtocol


Mercuric ion is an essential metal ion that plays an important role in a number of biological processes. Mercury ion is considered to be one of the most hazardous pollutants and highly dangerous materials. The high toxicity of Hg2+ is caused by its high affinity to the thiol groups in biological ligands such as proteins, DNA, and enzymes. When Hg2+ is absorbed in the human body from the environment, it induces aberrations in microtubules, ion channels, and mitochondria presumably and significant damage to the kidneys, heart, brain, stomach, intestines, central nervous system and immune systems. In addition, mercury accumulates through food chains or atmosphere in the ecological system, and has a relatively long atmospheric residence time because of its non-biodegradation. It has been intensively studies for a variety reasons, in particular, its environmental impacts and biological complications. However, there are very few commercial products that were developed for rapid detection mercuric ion. The highly selective and sensitive detection of mercury is of toxicological and environmental importance. Amplite® Fluorimetric Mercuric Ion Quantitation Kit offers a robust fluorescence-based assay for measuring mercury ion (Hg2+) with high selectivity. Mercury Lite™ 590 itself is nearly non-fluorescent, but generates more than 500-fold fluorescence enhancement upon binding Hg2+ ion. The fluorescence signal can be measured with a fluorescence microplate reader. With this kit, we were able to detect as low as 8 µM Hg2+ in a 100 µL reaction volume.

Platform


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare mercury working solution (50 µL)
  2. Add mercury (II) standard or test samples (50 µL)
  3. Incubate at room temperature for 20 - 30 minutes
  4. 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

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

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

Mercury (II) standard

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. 

BLBLTSTS
M1M1......
M2M2......
M3M3  
M4M4  
M5M5  
M6M6  
M7M6  

Table 2. Reagent composition for each well.

WellVolumeReagent
M1 - M750 µLSerial Dilutions (7.8 to 500 µM)
BL50 µLAssay Buffer
TS50 µLtest sample
  1. 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.

  2. 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.

  3. Incubate the reaction at room temperature for 20 - 30 minutes, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm, cutoff 570 nm.

Product Family


NameExcitation (nm)Emission (nm)
Amplite® Fluorimetric Zinc Ion Quantitation Kit493516

Images


References


View all 11 references: Citation Explorer
Coordination of mercury (II) to gold nanoparticle associated nitrotriazole towards sensitive colorimetric detection of mercuric ion with a tunable dynamic range
Authors: Chen, Xiaojun and Zu, Yanbing and Xie, Hong and Kemas, Aurino Muhammad and Gao, Zhiqiang
Journal: Analyst (2011): 1690--1696
Sequence and analysis of a plasmid-encoded mercury resistance operon from Mycobacterium marinum identifies MerH, a new mercuric ion transporter
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
Is the cytoplasmic loop of MerT, the mercuric ion transport protein, involved in mercury transfer to the mercuric reductase?
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
MerF is a mercury transport protein: different structures but a common mechanism for mercuric ion transporters?
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
Overexpression of MerT, the mercuric ion transport protein of transposon Tn501, and genetic selection of mercury hypersensitivity mutations
Authors: Hobman, Jonathan L and Brown, Nigel L
Journal: Molecular and General Genetics MGG (1996): 129--134
Heat sensitivity of mercuric ion and organomercurial degrading enzymes of aquatic, mercury-resistant bacteria
Authors: Pahan, K and Ray, S and Gachhui, R and Chaudhuri, J and M, undefined and al, A
Journal: World Journal of Microbiology and Biotechnology (1993): 180--183
Uptake of metallic mercury and mercuric ion by human erythrocytes.
Authors: Ogata, M and Ishii, K and Meguro, T
Journal: Physiological chemistry and physics and medical NMR (1990): 135--140
Levels of metallic mercury and mercuric ion in the venous and arterial bloods of normal and acatalasemic mice following exposure to mercury vapor.
Authors: Aikoh, H and Ogata, M
Journal: Physiological chemistry and physics and medical NMR (1988): 177--181