title: Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation.
abstract: Protein phosphorylation has traditionally been detected by radioisotope phosphate labeling of proteins with radioactive ATP. Several nonradioactive assays with phosphorylation site-specific antibodies are now available for the analysis of phosphorylation status at target sites. However, due to their high specificity, these antibodies they cannot be used to detect unidentified phosphorylation sites. Recently, Phos-tag technology has been developed to overcome the disadvantages and limitations of phosphospecific antibodies. Phos-tag and its derivatives conjugated to biotin, acrylamide, or agarose, form alkoxide-bridged dinuclear metal complexes, which can capture phosphate monoester dianions bound to serine, threonine, and tyrosine residues, in an amino acid sequence-independent manner. Here, we describe our method, which is based on in vitro kinase assay and Western blotting analysis using biotinylated Phos-tag and horseradish peroxidase-conjugated streptavidin, to determine the sites of TRPC6 (transient receptor potential canonical 6) channel phosphorylated by protein kinase A.
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Hokkaido, 060-8638, Japan. horinouc@med.hokudai.ac.jp.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Horinouchi
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Hokkaido, 060-8638, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Terada
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Hokkaido, 060-8638, Japan.
authors.forename: Tsunehito
authors.initials: T
authors.lastname: Higashi
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Hokkaido, 060-8638, Japan.
authors.forename: Soichi
authors.initials: S
authors.lastname: Miwa
mesh_terms: D015153:Blotting, Western / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism
chemical_list: D010750:Phosphoproteins; D010770:Phosphotransferases
keywords: Biotin; Horseradish peroxidase-conjugated streptavidin; In vitro kinase assay; Phos-tag; Protein kinase A; Protein phosphorylation; Western blotting