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Detection of protein‐protein interactions and a group of immunoglobulin G‐associated minor proteins in human plasma by nondenaturing and denaturing two‐dimensional gel electrophoresis
Author(s) -
Manabe Takashi,
Yamaguchi Nao,
Mukai Jun,
Hamada Osamu,
Tani Osamu
Publication year - 2003
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200300401
Subject(s) - isoelectric focusing , chemistry , gel electrophoresis , isoelectric point , difference gel electrophoresis , agarose , chromatography , electrophoresis , gel electrophoresis of proteins , polyacrylamide gel electrophoresis , two dimensional gel electrophoresis , biochemistry , proteomics , enzyme , gene
The dissociation of noncovalently associated protein‐protein complexes in human plasma was examined by comparing two‐dimensional gel electrophoresis (2‐DE) patterns obtained in two different electrophoretic conditions. A type I 2‐DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2‐DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro‐sized gels (internal diameter(id) 1.3×35 mm polyacrylamide IEF gels and 38×38×1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4×160 mm agarose IEF gels and 160×120×2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2‐D gels, newly appeared on type II large‐sized 2‐D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5–7.5 and relative molecular mass 8–45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2‐D gels. These minor spots could be concentrated on type II 2‐D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.