Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation
2016
Protein phosphorylationhas 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 potentialcanonical 6) channel phosphorylated by protein kinase A.
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