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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
Author(s) -
Masarik Michal,
Gumulec Jaromir,
Sztalmachova Marketa,
Hlavna Marian,
Babula Petr,
Krizkova Sona,
Ryvolova Marketa,
Jurajda Michal,
Sochor Jiri,
Adam Vojtech,
Kizek Rene
Publication year - 2011
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201100301
Subject(s) - metallothionein , cadmium , chemistry , zinc , lysis , metal ions in aqueous solution , copper , paramagnetism , cell culture , metal , chromatography , biochemistry , nuclear chemistry , biology , physics , organic chemistry , quantum mechanics , genetics
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.