Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose.
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 clearly separated as up-shifted migration bands in a 3% w/v polyacrylamide gel containing 20 microM 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).
Kinoshita
Eiji
E
Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8553, Japan. kinoeiji@hiroshima-u.ac.jp
Kinoshita-Kikuta
Emiko
E
Ujihara
Hiromi
H
Koike
Tohru
T
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry*; D012685:Sepharose / Q000032:analysis* / Q000737:chemistry*
D011506:Proteins; D012685:Sepharose