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 μM 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).