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Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes.
Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes.
- Source :
-
Electrophoresis [Electrophoresis] 2009 Feb; Vol. 30 (3), pp. 550-9. - Publication Year :
- 2009
-
Abstract
- 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 (Mn(2+)-Phos-tag SDS-PAGE). The first 2-D procedure coupling urea-PAGE and Mn(2+)-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 Mn(2+)-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 Mn(2+)-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 Mn(2+)-Phos-tag SDS-PAGE.
Details
- Language :
- English
- ISSN :
- 1522-2683
- Volume :
- 30
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Electrophoresis
- Publication Type :
- Academic Journal
- Accession number :
- 19156764
- Full Text :
- https://doi.org/10.1002/elps.200800386