This product includes 500 ul of 100 x NZA dye and 25 ul of 1 mM ZnCl2. It is for 1000 assays using 96-well plates. Cuvettes may also be used for measurements.
Zinc (Zn++) is an essential metal ion in biological systems. Zinc is a cofactor of hundreds of enzymes and plays important roles in signal transduction, gene expression, regulation of apoptosis, synaptic plasticity and prostate gland function. Zinc deficiency is associated with malabsorption syndrome, chronic GI, liver disease, diabetes, renal disease, sickle cell disease, anorexia nervosa, and HIV infection.
The NanoMolar Zinc Assay Kit is for measurement of submicromolar concentrations of zinc (0.1 µM – 2 µM). The assay is based on the principle that binding the fluorescence dye NZA selectively with Zinc results in increase of the fluorescence intensity (emission 535 nm, excitation 485 nm). The assay is compatible with regular buffers with different metal ions including 10 M Mg2+, Ca2+, Cu2+ , Mn2+, Al3+, Fe3+, Ag+ , Co2+ and Ni2+. Chelators such EDTA and thiol compounds bind zinc and should be avoided in the assay. The assay kit can be used for high-throughput measurements of zinc concentrations in biochemical assay reactions associated with zinc metabolism or environmental water samples.
The NanoMolar Zinc Assay Kit (catalog number NZA1000) includes 500 µl of 100 x NZA dye and 25 µl of 1 mM ZnCl2. It is for 1000 assays using 96-well plates. Cuvettes may also be used for measurements.
Concentration measurement
Metal ions
Reference
Dandley E. C. et al, Atomic layer deposition coating of carbon nanotubes with zinc oxide causes acute phase immune responses in human monocytes in vitro and in mice after pulmonary exposure. Particle and Fibre Toxicology, Vol.13: 29 (2016).
ASSAY PROTOCOL
The following assay protocol is based on using a 96-well plate for the measurement. The sample volume is 100 µl and the final assay volume is 150 µl. For 384-well plate assays, the sample volume is 60 µl and the final assay volume is 90 µl. For assays using cuvette, the sample volume is 800 µl and the final assay volume is 1200 µl.
STANDARD CURVE
1. Sample preparation: Prepare 100 µl of ZnCl2 solutions in the wells of a black 96-well plate with a two-fold serial dilution from 0.005 mM to zero in a 10 mM Tris-HCl or HEPES buffer, pH 7.4. Dilute the 100 x NZA dye 100-fold with water to make the 1 x NZA dye.
2. Detection: Mix 50 µl of 1 x NZA dye with 100 µl of the ZnCl2 solutions for 5 min and read the fluorescence at 535 nm (excitation at 485 nm).
3. Data Analysis: Plot the fluorescence intensity Fc and the zinc concentration [Zn] to generate the linear standard curve.
Fc = a [Zn] + Fo
Where the Fc values are from experimental data, the a and Fo values are from the linear fitting between the Fc values and the zinc concentrations.
UNKNOWN SAMPLES
Follow the same procedure to measure the fluorescence intensity Fc values from the unknown samples. Calculate the zinc concentrations in the unknown samples using the Fc values from the unknown samples and the a and Fo values from the standard curve.