Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes.
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.
Kinoshita
Eiji
E
Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. kinoeiji@hiroshima-u.ac.jp
Kinoshita-Kikuta
Emiko
E
Matsubara
Mamoru
M
Aoki
Yuri
Y
Ohie
Shiori
S
Mouri
Yuka
Y
Koike
Tohru
T
D000345:Affinity Labels; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D010750:Phosphoproteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D016875:tau Proteins / Q000378:metabolism
D000345:Affinity Labels; C054369:MAPT protein, human; D010750:Phosphoproteins; D016875:tau Proteins