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

Abstract

We provide a standard phosphate-affinity SDS-PAGE (Mn2+–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 Mn2+–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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Figure 1: Phosphate-affinity Mn2+-Phos-tag SDS-PAGE for the mobility-shift detection of phosphoproteins.
Figure 2: Schematic representation of the relationship between the degree of migration (Rf value) and molecular weight for several proteins (200–350 kDa) in 2.7–3.5% (wt/vol) polyacrylamide slab gels.
Figure 3: Separation and detection of the phosphoisotypes of mTOR.
Figure 4: Phosphorylation of DNA damage signaling-related proteins in HeLa cells treated with actinomycin D.

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Acknowledgements

This work was supported by Grants-in-Aid for Scientific Research (B) (19390011) and (C) (19590040) from the Japan Society of the Promotion of Science (JSPS), a Grant-in-Aid for Young Scientists (B) (20790036) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) and a research grant from the Takeda Science Foundation.

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E.K., E.K.-K. and T.K. conceived, designed and performed the experiments and wrote the paper.

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Correspondence to Eiji Kinoshita.

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Kinoshita, E., Kinoshita-Kikuta, E. & Koike, T. Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE. Nat Protoc 4, 1513–1521 (2009). https://doi.org/10.1038/nprot.2009.154

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