Detection of phosphorylation by western blotting is an important procedure to elucidate molecular mechanisms in signal transduction pathways involving kinases and phosphatases. Anti-phospho-tyrosine monoclonal antibodies have been widely used because they react with plethora of proteins containing phosphorylated tyrosine residues. In contrast, the monoclonal antibodies against phospho-serine or threonine residues are unpopular, since their affinity and specificity are less than optimal. To achieve precise characterization of signaling events, it is desirable to raise a good anti-phospho-site-specific antibody to clearly detect phosphorylated species. However, raising this type of antibody is costly and time-consuming, and sometimes results in failure.
Use of Phos-tag may provide an alternative method to detect phosphorylated proteins. Phos-tag is a dinuclear metal complex that acts as a novel phosphate-binding tag. Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. Phosphorylated proteins can be detected as slower migrating species by electrophoresis and western blotting using PAGE gel containing appropriate amount of Phos-tag acrylamide.
Previously Phos-tag acrylamide has been used in 20-100 μM concentrations. We have now found that lower concentrations (3.5-5 μM) of Phos-tag can dramatically improve the separation between phosphorylated and non-phosphorylated species of a protein with larger molecular mass (~150 kDa). Here we describe a detailed protocol that includes tips to ensure easier application of this methodology. The procedure should be carried out as in the standard SDS-PAGE unless stated otherwise.