Amplite® Colorimetric 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® Colorimetric Zinc Quantitation Kit provides a simple method for detecting zinc concentration in biological samples using our proprietary Zn-620™, in which Zinc binds to the probe with the enhanced absorption around 620 nm. The Zinc probe exhibits a large increase in 620 nm absorption in response to Zn2+ (>100 folds). Our kit formulation has enhanced Zn2+-specificity with little responses to other metals, e.g., Ca2+ and Mg2+. The assay can be used with biological samples such as serum, plasma, and urine with detection sensitivity at 1 µM. Our Amplite® Fluorimetric Zinc Ion Quantitation Kit (#19000) is even more sensitive, and can be used for detecting as low as 0.1 uM zinc ion.
Example protocol
AT A GLANCE
Protocol Summary
- Prepare Zn2+ Standards or test samples (50 µL)
- Add Zinc working solution (50 µL)
- Incubate at room temperature for 5 - 10 minutes
- Read absorbance ratio of A610nm/A470nm
Important Note
Thaw all kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTIONS
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
ZnCl2 standard solution (1 mM)
Add 10 µL of 100 mM ZnCl2 Standard solution (Component C) into 990 µL Assay Buffer (Component B) to get 1 mM ZnCl2 standard solution.
PREPARATION OF STANDARD SOLUTIONS
For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/19001
https://www.aatbio.com/tools/serial-dilution/19001
ZnCl2 standard
Add 100 µL of 1 mM ZnCl2 standard solution to 900 µL Assay Buffer (Component B) to get 100 µM ZnCl2 standard solution (Zn1). Take 100 µM ZnCl2 standard solution (Zn1) and perform 1:2 serial dilutions in Assay Buffer (Component B) to get serially diluted ZnCl2 standards (Zn7 - Zn2).PREPARATION OF WORKING SOLUTION
Add 25 μL of Zn-620™ (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 clear bottom 96-well microplate. Zn= Zinc Standards (Zn1 - Zn7, 100 to 1.56 µM), BL=Blank Control, TS=Test Samples.
BL | BL | TS | TS |
Zn1 | Zn1 | ... | ... |
Zn2 | Zn2 | ... | ... |
Zn3 | Zn3 | ||
Zn4 | Zn4 | ||
Zn5 | Zn5 | ||
Zn6 | Zn6 | ||
Zn7 | Zn7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
Zn1 - Zn7 | 50 µL | Serial Dilutions (100 to 1.56 µM) |
BL | 50 µL | Assay Buffer |
TS | 50 µL | test sample |
- Dilute the test sample to 1.56 - 100 µM range with Assay Buffer (Component B).
- Prepare ZnCl2 standards (Zn), 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 Zn working solution to 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 Zn working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction for 5 - 10 minutes at room temperature, protected from light.
- Monitor the absorbance ratio increase with a absorbance plate reader at A610nm/A470nm.
Product family
Name | Excitation (nm) | Emission (nm) |
Amplite® Fluorimetric Zinc Ion Quantitation Kit | 493 | 516 |
Citations
View all 4 citations: Citation Explorer
Growth Inhibition of Gram-Positive and Gram-Negative Bacteria by Zinc Oxide Hedgehog Particles
Authors: Rutherford, David and J{\'\i}ra, Jaroslav and Kol{\'a}{\v{r}}ov{\'a}, Kate{\v{r}}ina and Matol{\'\i}nov{\'a}, Iva and Mi{\v{c}}ov{\'a}, J{\'u}lia and Reme{\v{s}}, Zdenek and Rezek, Bohuslav
Journal: International Journal of Nanomedicine (2021): 3541
Authors: Rutherford, David and J{\'\i}ra, Jaroslav and Kol{\'a}{\v{r}}ov{\'a}, Kate{\v{r}}ina and Matol{\'\i}nov{\'a}, Iva and Mi{\v{c}}ov{\'a}, J{\'u}lia and Reme{\v{s}}, Zdenek and Rezek, Bohuslav
Journal: International Journal of Nanomedicine (2021): 3541
Kinetic Effects of Heat Stress on Olfaction: A Thermodynamic Evaluation of Electrical Responses to Odorants in Olfactory Epithelia
Authors: Hagerty, Samantha
Journal: (2018)
Authors: Hagerty, Samantha
Journal: (2018)
The role of zinc nanoparticles in the initial events of olfaction, their characterization, preservation, and microenvironmental influence.
Authors: Singletary, Melissa
Journal: (2018)
Authors: Singletary, Melissa
Journal: (2018)
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
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
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
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
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
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
Authors: Nowakowski AB, Petering DH.
Journal: Inorg Chem (2011): 10124
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