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

• Published in: Nature protocols Vol. 4; no. 10; pp. 1513 - 1521
• Main Authors: Kinoshita, Eiji, Kinoshita-Kikuta, Emiko, Koike, Tohru
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-01-2009; Nature Publishing Group
• Subjects: Analytical Chemistry; Ataxia Telangiectasia Mutated Proteins; Biological Techniques; Cell cycle; Cell Cycle Proteins - chemistry; Computational Biology/Bioinformatics; DNA-Binding Proteins - chemistry; Electrophoresis; Electrophoresis, Polyacrylamide Gel - methods; Electrophoretic Mobility Shift Assay; Gel electrophoresis; HeLa Cells; Humans; Identification and classification; Intracellular Signaling Peptides and Proteins - chemistry; Kinases; Life Sciences; Mammals; Manganese; Methods; Microarrays; Monoclonal antibodies; Organic Chemistry; Phosphoproteins; Phosphoproteins - analysis; Phosphoproteins - chemistry; Phosphoproteins - isolation & purification; Phosphorylation; Protein Kinases - chemistry; Protein-Serine-Threonine Kinases - chemistry; Proteins; Protocol; TOR Serine-Threonine Kinases; Tumor Suppressor p53-Binding Protein 1; Tumor Suppressor Proteins - chemistry; Zinc; Japan
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2009.154

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 (∼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.