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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.

Authors :
Kinoshita E
Kinoshita-Kikuta E
Matsubara M
Aoki Y
Ohie S
Mouri Y
Koike T
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