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AAT Bioquest

Amplite® Fluorimetric Zinc Ion Quantitation Kit

Zinc is an essential trace mineral element that plays an important role in a number of biological processes. It is an essential factor required for many enzymes, protein structures, and control of genetic expression. Zinc status also affects basic processes of cell division, growth, differentiation, development, and aging. Clinical signs of zinc deficiency include acrodermatitis, low immunity, diarrhea, poor healing, stunting, hypogonadism, fetal growth failure, teratology and abortion. Simple, direct and automation-ready procedures for measuring are highly desirable in research and drug discovery. AAT Bioquest's Amplite® Fluorimetric Zinc Quantitation Kit provides a simple method for detecting zinc concentration in biological samples using our proprietary Metaltel; Zn 520, in which Zinc binds to the probe with enhanced fluorescence. The Zinc probe exhibits a large increase in fluorescence in response to Zn2+ (greater than 200~300 folds). It has high Zn2+-specificity with little responses to other metals, including Ca2+, Mg2+, Mn2+, and Cu2+. The assay can be used with biological samples such as serum, plasma, and urine with detection sensitivity at 0.2 µM (13 ng/mL).

Example protocol

AT A GLANCE

Protocol summary

  1. Test samples (50 µL) or Zn2+ Standard
  2. Add Zinc Probe Reagent (50 µL)
  3. Incubate at room temperature for 5 - 10 minutes
  4. Read fluorescence at Ex/Em= 485/525 nm

Important notes
Thaw all the 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. Zinc standard solution (1 mM):
Add 10 µL of 100 mM ZnCl2 Standard (Component C) into 990 µL Assay Buffer (Component B) to get 1 mM ZnCl2 standard solution.

PREPARATION OF STANDARD SOLUTION

ZnCl2 standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/19000

Add 100 µL of 1 mM Zinc standard solution to 900 µL Assay Buffer (Component B) to get 100 µM ZnCl2 standard solution (Zn7). Then take the 100 µM ZnCl2 standard solution to perform 1:3 serial dilutions to get serially diluted ZnCl2 standards (Zn6 - Zn1).

PREPARATION OF WORKING SOLUTION

Add 25 µL of Metal Fluor™ Zn 520 (Component A) into 5 mL Assay Buffer (Component B) to make Zn working solution.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of ZnCl2 standards and test samples in a solid black 96-well microplate. Zn = Zinc standard (Zn1 - Zn7, 0.1 to 100 µM); BL = blank control; TS = test sample.

BLBLTSTS
Zn1Zn1......
Zn2Zn2......
Zn3Zn3  
Zn4Zn4  
Zn5Zn5  
Zn6Zn6  
Zn7Zn7  

Table 2. Reagent compotition for each well.

WellVolumeReagent
Zn1 - Zn750 µLserial dilution (0.1 to 100 µM)
BL50 µLAssay Buffer (Component B)
TS50 µLsample
  1. Dilute the test sample to a 5 - 100 µM range with Assay Buffer (Component B).

  2. Prepare ZnCl2 standards (Zn), blank controls (BL), and test samples (TS) 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.

  3. Add 50 µL of Zinc working solution into each well of ZnCl2 standard, blank control, and test samples to make the total ZnCl2 assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Zinc assay buffer into each well instead, for a total volume of 50 µL/well.

  4. Incubate the reaction for 5 - 10 minutes at room temperature, protected from light.

  5. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 485/525 nm.

Spectrum

Citations

View all 3 citations: Citation Explorer
ZIP1+ fibroblasts protect lung cancer against chemotherapy via connexin-43 mediated intercellular Zn2+ transfer
Authors: Ni, Chen and Lou, Xiaohan and Yao, Xiaohan and Wang, Linlin and Wan, Jiajia and Duan, Xixi and Liang, Jialu and Zhang, Kaili and Yang, Yuanyuan and Zhang, Li and others,
Journal: Nature communications (2022): 1--20
Hatching enzymes disrupt aberrant gonadal degeneration by the autophagy/apoptosis cell fate decision
Authors: Chakraborty, Tapas and Mohapatra, Sipra and Tobayama, Megumi and Ohta, Kayoko and Ryu, Yong-Woon and Kazeto, Yukinori and Ohta, Kohei and Zhou, Linyan and Nagahama, Yoshitaka and Matsubara, Takahiro
Journal: Scientific reports (2017): 1--12
After oxidation, zinc nanoparticles lose their ability to enhance responses to odorants
Authors: Hagerty, Samantha and Daniels, Yasmine and Singletary, Melissa and Pustovyy, Oleg and Globa, Ludmila and MacCrehan, William A and Muramoto, Shin and Stan, Gheorghe and Lau, June W and Morrison, Edward E and others, undefined
Journal: BioMetals (2016): 1--14

References

View all 46 references: Citation Explorer
Reaction of metal-binding ligands with the zinc proteome: zinc sensors and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine
Authors: Meeusen JW, Nowakowski A, Petering DH.
Journal: Inorg Chem (2012): 3625
EAAC1 gene deletion alters zinc homeostasis and enhances cortical neuronal injury after transient cerebral ischemia in mice
Authors: Jang BG, Won SJ, Kim JH, Choi BY, Lee MW, Sohn M, Song HK, Suh SW.
Journal: J Trace Elem Med Biol (2012): 85
TSQ (6-methoxy-8-p-toluenesulfonamido-quinoline), a common fluorescent sensor for cellular zinc, images zinc proteins
Authors: Meeusen JW, Tomasiewicz H, Nowakowski A, Petering DH.
Journal: Inorg Chem (2011): 7563
Dependence of the histofluorescently reactive zinc pool on zinc transporter-3 in the normal brain
Authors: Lee JY, Kim JS, Byun HR, Palmiter RD, Koh JY.
Journal: Brain Res (2011): 12
Reactions of the fluorescent sensor, Zinquin, with the zinc-proteome: adduct formation and ligand substitution
Authors: Nowakowski AB, Petering DH.
Journal: Inorg Chem (2011): 10124
Page updated on December 4, 2024

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Catalog Number19000
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Spectral properties

Excitation (nm)

493

Emission (nm)

516

Storage, safety and handling

H-phraseH303, H313, H340
Hazard symbolT
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R68
UNSPSC41116134

Platform

Fluorescence microplate reader

Excitation485 nm
Emission525 nm
Cutoff515 nm
Recommended plateSolid black

Components

Zinc Chloride dose response was measured on a 96-well black plate with the Amplite® Fluorimetric Zinc Ion Quantitation Kit.
Zinc Chloride dose response was measured on a 96-well black plate with the Amplite® Fluorimetric Zinc Ion Quantitation Kit.
Zinc Chloride dose response was measured on a 96-well black plate with the Amplite® Fluorimetric Zinc Ion Quantitation Kit.