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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Ataxia Telangiectasia Mutated Proteins
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Cell Cycle Proteins / chemistry
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DNA-Binding Proteins / chemistry
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Electrophoresis, Polyacrylamide Gel / methods*
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Electrophoretic Mobility Shift Assay
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HeLa Cells
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Humans
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Intracellular Signaling Peptides and Proteins / chemistry
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Manganese
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Phosphoproteins / analysis
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Phosphoproteins / chemistry*
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Phosphoproteins / isolation & purification
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Phosphorylation
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Protein Kinases / chemistry
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Protein Serine-Threonine Kinases / chemistry
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TOR Serine-Threonine Kinases
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Tumor Suppressor Proteins / chemistry
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Tumor Suppressor p53-Binding Protein 1
Substances
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Cell Cycle Proteins
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DNA-Binding Proteins
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Intracellular Signaling Peptides and Proteins
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Phosphoproteins
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TP53BP1 protein, human
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Tumor Suppressor Proteins
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Tumor Suppressor p53-Binding Protein 1
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Manganese
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Protein Kinases
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MTOR protein, human
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ATM protein, human
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Ataxia Telangiectasia Mutated Proteins
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Protein Serine-Threonine Kinases
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TOR Serine-Threonine Kinases