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Nat Protoc. 2009;4(10):1513-21. doi: 10.1038/nprot.2009.154. Epub 2009 Sep 24.

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

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Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima, Japan.


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.

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