QuantiChromTM Zinc Assay Kit (DIZN-250)

Quantitative Colorimetric Zinc Determination at 425nm

Zinc is an essential trace element and plays many key roles in metabolism. It is required for the activity of more than 300 enzymes, the structure of many proteins, and control of genetic expression. Zinc status affects basic processes of cell division, growth, differentiation, development, performance and aging through its requirement for synthesis and repair of DNA, RNA and protein. The common causes of zinc deficiency are low dietary intakes and low bioavailability. Clinical signs of zinc deficiency include acrodermatitis, low immunity, diarrhea, poor healing, stunting, hypogonadism, fetal growth failure, teratology and abortion. Zinc deficiency1 has now been recognized to be associated with many diseases such as malabsorption syndrome, chronic liver disease, chronic renal disease, sickle cell disease, diabetes, malignancy, and other chronic illnesses. Simple, direct and automation-ready procedures for measuring zinc concentration in biological samples are highly desirable in Research and Drug Discovery. BioAssay Systems' zinc assay kit is designed to measure zinc directly in biological samples without any pretreatment. The present method utilizes a chromogen that forms a colored complex specifically with zinc. The intensity of the color, measured at 425 nm, is directly proportional to the zinc concentration in the sample.

Simple and high-throughput. The procedure involves addition of a single working reagent and incubation for 30 min. Can be readily automated as a high-throughput assay for thousands of samples per day.

Improved reagent stability and versatility. The optimized formulation has greatly enhanced reagent and signal stability. Cuvette or 96-well plate assay formats possible.

Low interference in biological samples. No pretreatments are needed. Assays can be directly performed on raw biological samples, i.e. in the presence of lipid and protein.

Drug Discovery/Pharmacology: effects of drugs on zinc metabolism.

Environment: zinc determination in waste water, soil etc.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

Sample preparation: serum and plasma samples should be clear and free of turbidity or precipitates. If present, precipitates should be removed by filtration or centrifugation on a table centrifuge. Prior to assay, dilute serum or plasma samples 5-fold (n = 5) in deionized water.

Reagent preparation: equilibrate all reagents to room temperature. Vortex Reagents B and C before assay. Prepare enough Working Reagent: for each assay well, mix 200 μL Reagent A, 4 μL Reagent B and 4 μL reagent C. For each test, run a standard (10 μM Zn2+), water blank, sample and sample blank (sample + EDTA). EDTA chelates Zn2+ in the sample, prevents Zn2+-Dye interaction and is used to prepare the sample blank.

Procedure using 96-well plate:

Transfer 200 μL water, standard, sample and sample blank (200 μL Sample + 8 μL EDTA) to appropriately labeled tubes. Add 800 μL working reagent and tap lightly to mix. Incubate 30 min and read optical density at 425 nm.

GENERAL CONSIDERATIONS

Because the shift in the peak wavelength (from 413 nm to 425 nm) is very small, the color change is not visually evident. Physiological concentrations of other metal ions do not interfere. Zn2+ chelators (e.g. EDTA, EGTA) should be avoided during sample preparation.

CALCULATION

Conversions: 1 μM zinc equals 6.5 μg/dL or 0.065 ppm (65 ppt).

Pipeting devices and accessories. Clear bottom 96-well plates (e.g. Corning Costar) and plate reader, or cuvettes and spectrophotometer for measuring OD 425nm.

[1]. Prasad. AS (2003) Zinc deficiency. BMJ 326:409-410.

[2]. Neal DE Jr, Kaack MB, Fussell EN, Roberts JA. (1993) Changes in seminal fluid zinc during experimental prostatitis. Urol Res. 21(1): 71-74.