☆ 4.5 Article

Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose

PROTEOMICS (2009)

期刊

PROTEOMICS

卷 9, 期 16, 页码 4098-4101

出版社

WILEY

DOI: 10.1002/pmic.200900020

关键词

High-molecular-mass proteins; Phosphate-affinity SDS-PAGE; Phosphorylation; Phos-tag; Technology; Western blotting

类别

Biochemical Research Methods Biochemistry & Molecular Biology

资金

Japan Society for the Promotion of Science and for Young Scientists [19390011, 19590040]
Ministry of Education, Culture, Sports, Science, and Technology [20790036]
Grants-in-Aid for Scientific Research [19390011, 20790036, 19590040] Funding Source: KAKEN

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摘要

We describe a novel technique of phosphate-affinity SDS-PAGE using Phos-tag to analyze large phosphoproteins with molecular masses of more than 200 kDa. The protein phosphoisotypes were dearly separated as up-shifted migration bands in a 3% w/v polyacrylamide gel containing 20 mu M Phos-tag and 0.5% w/v agarose. In subsequent immunoblotting, the procedure permitted the determination of the phosphoisotypes of high-molecular-mass proteins, such as mTOR (289 kDa), ATM kinase (350 kDa), and 53BP1 (213 kDa).

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