AbstractWe provide a standard phosphate-affinity SDS-PAGE (Mn2+鈥揚hos-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 (鈭?/span>
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+鈥揚hos-tag SDS-PAGE gel. The procedure from the beginning of gel preparation to the end of electrophoresis requires about 4 h in this protocol. Subscription info for Chinese customersWe have a dedicated website for our Chinese customers. Please go to naturechina.com to subscribe to this journal.Go to naturechina.comRent or Buy articleGet time limited or full article access on ReadCube.from$8.99Rent or BuyAll prices are NET prices. 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Chem. 278, 19579鈥?9582 (2003).CAS聽 Article聽Google Scholar聽 Download referencesAcknowledgementsThis 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.Author informationAffiliationsDepartment of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima, JapanEiji Kinoshita,聽Emiko Kinoshita-Kikuta聽 聽Tohru KoikeAuthorsEiji KinoshitaView author publicationsYou can also search for this author in PubMed聽Google ScholarEmiko Kinoshita-KikutaView author publicationsYou can also search for this author in PubMed聽Google ScholarTohru KoikeView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsE.K., E.K.-K. and T.K. conceived, designed and performed the experiments and wrote the paper.Corresponding authorCorrespondence to Eiji Kinoshita.Supplementary information Supplementary Figure 1 (PDF 112 kb)Rights and permissionsReprints and PermissionsAbout this articleCite this articleKinoshita, E., Kinoshita-Kikuta, E. Koike, T. Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE. Nat Protoc 4, 1513鈥?521 (2009). https://doi.org/10.1038/nprot.2009.154Download citationPublished: 24 September 2009Issue Date: October 2009DOI: https://doi.org/10.1038/nprot.2009.154 Thorsten Stehlik, Marco Kremp, J枚rg Kahnt, Michael B枚lker Johannes Freitag Nature Communications (2020) Xia Yu, Xiaopan Gao, Kaixiang Zhu, Han Yin, Xujian Mao, Justyna Aleksandra Wojdyla, Bo Qin, Hairong Huang, Meitian Wang, Yi-Cheng Sun Sheng Cui Communications Biology (2020) Mami Yasukawa, Yoshinari Ando, Taro Yamashita, Yoko Matsuda, Shisako Shoji, Masaki Suimye Morioka, Hideya Kawaji, Kumiko Shiozawa, Mitsuhiro Machitani, Takaya Abe, Shinji Yamada, Mika K. 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