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Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE
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Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE
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Journal Article
Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE
2009
Overview
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 (∼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.
Publisher
Nature Publishing Group UK,Nature Publishing Group
Subject
/ Ataxia Telangiectasia Mutated Proteins
/ Cell Cycle Proteins - chemistry
/ Computational Biology/Bioinformatics
/ DNA-Binding Proteins - chemistry
/ Electrophoresis, Polyacrylamide Gel - methods
/ Electrophoretic Mobility Shift Assay
/ Humans
/ Identification and classification
/ Intracellular Signaling Peptides and Proteins - chemistry
/ Kinases
/ Mammals
/ Methods
/ Phosphoproteins - isolation & purification
/ Protein-Serine-Threonine Kinases - chemistry
/ Proteins
/ Protocol
/ TOR Serine-Threonine Kinases
/ Tumor Suppressor p53-Binding Protein 1
/ Tumor Suppressor Proteins - chemistry
/ Zinc
