Isolation of metallothionein from cells derived from aggressive form of high-grade prostate carcinoma using paramagnetic antibody-modified microbeads off-line coupled with electrochemical and electrophoretic analysis
Language English Country Germany Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Cell Extracts chemistry MeSH
- Time Factors MeSH
- Electric Impedance MeSH
- Electrophoresis, Polyacrylamide Gel methods MeSH
- Microscopy, Fluorescence MeSH
- Immunoglobulin G chemistry metabolism MeSH
- Immunoprecipitation methods MeSH
- Hydrogen-Ion Concentration MeSH
- Humans MeSH
- Linear Models MeSH
- Magnets * MeSH
- Metallothionein isolation & purification metabolism MeSH
- Microspheres MeSH
- Antibodies, Monoclonal chemistry MeSH
- Cell Line, Tumor MeSH
- Prostatic Neoplasms chemistry metabolism pathology MeSH
- Temperature MeSH
- Metals, Heavy MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Cell Extracts MeSH
- Immunoglobulin G MeSH
- Metallothionein MeSH
- Antibodies, Monoclonal MeSH
- Metals, Heavy MeSH
Prostate cancer with altered zinc(II) cell metabolism is the second most frequently diagnosed cancer in developed countries. The alterations of zinc(II) metabolism can influence metabolism of other metal ions and can also be associated with the expression and translation of metal-binding proteins including metallothioneins. The aim of this article was to optimize immunoseparation protocol based on paramagnetic beads conjugated with protein G for the isolation of metallothionein. Isolated metallothionein was determined by differential pulse voltammetry Brdicka reaction and SDS-PAGE. Optimal conditions: antigen-binding time - 60 min, temperature - 70°C, and buffer composition and pH - acetate buffer, pH 4.3, were determined. Under the optimized conditions, lysates from 22Rv1 prostate cancer cells treated with various concentrations of cadmium(II) and copper(II) ions were analyzed. We observed strong correlation in all experimental groups and all lysate types (r>0.83 at p<0.041) between metallothionein concentration related to viability and concentration of copper(II) ions and cadmium(II) ions in medium. Moreover, the results were compared with standard sample preparation as heat treatment and SDS-PAGE analysis.
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