期刊
ELECTROPHORESIS
卷 30, 期 3, 页码 550-559出版社
WILEY-BLACKWELL
DOI: 10.1002/elps.200800386
关键词
2-D affinity gel electrophoresis; Phosphoprotein; Phosphorylation; Phos-tag
资金
- Japan Society for the Promotion of Science [19390011, 19590040]
- Ministry of Education, Culture, Sports, Science, and Technology, [20790036]
- Iketani Science and Technology Foundation
- Takeda Science Foundation
- Grants-in-Aid for Scientific Research [20790036, 19590040, 19390011] Funding Source: KAKEN
Herein, we describe three kinds of 2-DE using phosphate-affinity PAGE for the analysis of phosphoprotein isotypes. The first dimension is a urea-PAGE, IEF/NEPHGE, or SDS-PAGE, which are widely used. The second dimension is a phosphate-affinity SDS-PAGE using a phosphate-binding tag molecule, Phos-tag (Mn2+-Phos-tag SDS-PAGE). The first 2-D procedure coupling urea-PAGE and Mn2+-Phos-tag SDS-PAGE was applied to the separation of beta-casein phosphoisotypes. A typical protein sample containing multiple phosphoisotypes from beta-casein (with five phosphorylation sites) was prepared by partial dephosphorylation with alkaline phosphatase. The second procedure coupling IEF/NEPHGE and Mn2+-Phos-tag SDS-PAGE was applied to the separations of phosphoisotypes of caseins and in vitro kinase reaction products of Tau. The third procedure coupling normal SDS-PAGE and Mn2+-Phos-tag SDS-PAGE was applied to the separation of A431 cell lysates before and after stimulation with an epidermal growth factor. This procedure followed by immunoblotting with anti-mitogen-activated protein kinase(MAPK) and anti-Shc antibodies demonstrated the detection of phosphoisotypes in each protein isoform of MAPK1/2 (44 and 42 kDa) and Shc (66, 52, and 46 kDa) after the stimulation. By these novel 2-D procedures, the separations of phosphoprotein isotypes should be improved relative to those by current gel electrophoresis methods, including 1-D Mn2+-Phos-tag SDS-PAGE.
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