Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation.
Methods Mol Biol
; 1397: 267-277, 2016.
Article
in En
| MEDLINE
| ID: mdl-26676139
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Phosphoproteins
/
Blotting, Western
Limits:
Humans
Language:
En
Journal:
Methods Mol Biol
Journal subject:
BIOLOGIA MOLECULAR
Year:
2016
Document type:
Article
Affiliation country:
Japón