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

Nat Protoc. 2009;4(10):1513-21. doi: 10.1038/nprot.2009.154. Epub 2009 Sep 24.

Abstract

We provide a standard phosphate-affinity SDS-PAGE (Mn(2+)-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 ( approximately 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 Mn(2+)-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ataxia Telangiectasia Mutated Proteins
  • Cell Cycle Proteins / chemistry
  • DNA-Binding Proteins / chemistry
  • Electrophoresis, Polyacrylamide Gel / methods*
  • Electrophoretic Mobility Shift Assay
  • HeLa Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins / chemistry
  • Manganese
  • Phosphoproteins / analysis
  • Phosphoproteins / chemistry*
  • Phosphoproteins / isolation & purification
  • Phosphorylation
  • Protein Kinases / chemistry
  • Protein-Serine-Threonine Kinases / chemistry
  • TOR Serine-Threonine Kinases
  • Tumor Suppressor Proteins / chemistry
  • Tumor Suppressor p53-Binding Protein 1

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Intracellular Signaling Peptides and Proteins
  • Phosphoproteins
  • TP53BP1 protein, human
  • Tumor Suppressor Proteins
  • Tumor Suppressor p53-Binding Protein 1
  • Manganese
  • Protein Kinases
  • MTOR protein, human
  • TOR Serine-Threonine Kinases
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Protein-Serine-Threonine Kinases