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Immunoextraction of zinc proteins from human plasma using chicken yolk antibodies immobilized onto paramagnetic particles and their electrophoretic analysis
Author(s) -
Krizkova Sona,
Ryvolova Marketa,
Hynek David,
Eckschlager Tomas,
Hodek Petr,
Masarik Michal,
Adam Vojtech,
Kizek Rene
Publication year - 2012
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.201100638
Subject(s) - zinc , chemistry , chromatography , horseradish peroxidase , elution , electrophoresis , extraction (chemistry) , prussian blue , electrochemistry , biochemistry , enzyme , electrode , organic chemistry
Zinc(II) as the only transition metal lacking redox activity is an essential part of approximately 10% proteins as a cofactor of these proteins. Considering the fact that there are numerous zinc(II) containing proteins, proteomics and metallomics studies aimed on them require accurate methods for preparation of real biological samples prior to their subsequent analysis using 2DE and MS. For this purpose, we suggested a new method based on chicken anti‐zinc antibodies and magnetizable particles. Antibodies were covalently immobilized to the surface of paramagnetic beads activated with tosyl group. Binding of the antibody to the beads was confirmed by secondary anti‐chicken antibody conjugated with horseradish peroxidase. The immunoextraction conditions, such as concentration of the beads (6–18 μg/mL of the sample), time of immunoextraction (6–34 min), pH and composition of the elution buffer, and time of extraction (48–300 s) were optimized. Subsequently, zinc proteins were extracted from human plasma and total concentration of zinc was monitored by electrochemical detection in the extracts. Under optimal conditions it was possible to monitor the proteins and zinc removal from the sample by chip CE, SDS‐PAGE, and indirectly using electrochemistry.