Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE.

Abstract	We provide a standard phosphate-affinity SDS-PAGE (Mn(2+)-Phos-tag SDS-PAGE) protocol, in which Phos-tag is used to analyze large phosphoproteins with molecular masses of more than 200 kDa. A previous protocol required a long electrophoresis time of 12 h for separation of phosphoisotypes of large proteins ( approximately 150 kDa). This protocol, which uses a 3% (wt/vol) polyacrylamide gel strengthened with 0.5% (wt/vol) agarose, permits the separation of protein phosphoisotypes larger than 200 kDa within 2 h. In subsequent immunoblotting, phosphoisotypes of high-molecular-mass proteins, such as mammalian target of rapamycin (289 kDa), ataxia telangiectasia-mutated kinase (350 kDa) and p53-binding protein 1 (213 kDa), can be clearly detected as up-shifted migration bands on the improved Mn(2+)-Phos-tag SDS-PAGE gel. The procedure from the beginning of gel preparation to the end of electrophoresis requires about 4 h in this protocol.
Authors	Eiji Kinoshita, Emiko Kinoshita-Kikuta, Tohru Koike
Journal	Nature protocols (Nat Protoc) Vol. 4 Issue 10 Pg. 1513-21 ( 2009) ISSN: 1750-2799 [Electronic] England
PMID	19798084 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References	Cell Cycle Proteins DNA-Binding Proteins Intracellular Signaling Peptides and Proteins Phosphoproteins TP53BP1 protein, human Tumor Suppressor Proteins Tumor Suppressor p53-Binding Protein 1 Manganese Protein Kinases MTOR protein, human ATM protein, human Ataxia Telangiectasia Mutated Proteins Protein Serine-Threonine Kinases TOR Serine-Threonine Kinases
Topics	Ataxia Telangiectasia Mutated Proteins Cell Cycle Proteins (chemistry) DNA-Binding Proteins (chemistry) Electrophoresis, Polyacrylamide Gel (methods) Electrophoretic Mobility Shift Assay HeLa Cells Humans Intracellular Signaling Peptides and Proteins (chemistry) Manganese Phosphoproteins (analysis, chemistry, isolation & purification) Phosphorylation Protein Kinases (chemistry) Protein Serine-Threonine Kinases (chemistry) TOR Serine-Threonine Kinases Tumor Suppressor Proteins (chemistry) Tumor Suppressor p53-Binding Protein 1

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