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 β‐casein phosphoisotypes. A typical protein sample containing multiple phosphoisotypes from β‐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.