Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate‐affinity SDS‐PAGE

Abstract

Herein, we demonstrate the separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate‐affinity SDS‐PAGE. The phosphate‐affinity site is a polyacrylamide‐bound Phos‐tag that enables the mobility shift detection of phosphoproteins from their nonphosphorylated counterparts. As the first practical example of the separation, we characterized the monophosphorylated Tau isotypes by each of three tyrosine kinases, c‐Abl, MET, and Fyn. Each monophosphoisotype phosphorylated at the Tyr‐394, Tyr‐197, or Tyr‐18 was detected as three distinct migration bands. As a further application, we extended this technique to the mobility shift analysis of His and Asp phosphoisotypes in the Sinorhizobium meliloti FixL/FixJ two‐component system. FixL is autophosphorylated at the His‐285 with ATP, and the phosphate group is transferred to the Asp‐54 of FixJ and subsequently removed by the FixL phosphatase activity. Using this method, we first performed simultaneous detection of the phosphorylated and nonphosphorylated isotypes of FixL and FixJ generated in their phosphotransfer reaction in vitro. As a result, a monophosphoisotype of FixL containing the phosphorylated His residue was confirmed. As for FixJ, on the other hand, two monophosphoisotypes were detected as two distinct migration bands. One is a well‐known isotype phosphorylated at the Asp‐54. The other is a novel isotype phosphorylated at the His‐84.

Phosphorylation-Regulated Transitions in an Oligomeric State Control the Activity of the Sae2 DNA Repair Enzyme

“…Reaction products were separated by SDS-PAGE in the presence of Phos-tag reagent to accentuate the differences in charge induced by phosphorylation (33) and then analyzed by protein blotting for phospho-SQ/TQ residues and for total Sae2 (Fig. 4D).…”

mTORC1 Directly Phosphorylates and Regulates Human MAF1

“…To extend these observations, we generated permanent IMR90hTert cell lines expressing a MAF1-EGFP fusion protein. We used SDSpolyacrylamide gels containing Phos-tag to separate phosphorylated forms of the fusion protein (15,16) and visualized them by immunoblotting with anti-EGFP antibodies. As shown in the top panels of Fig.…”

Advances in Phos-tag-based methodologies for separation and detection of the phosphoproteome

“…Because this system is compatible with the equipment and reagents used for the conventional SDS-PAGE followed by general Western blotting analysis with a specific antibody against a certain target [27][28][29][30][31], many researchers have used it in the intervening years and have reported valuable findings related to protein phosphorylation modifications not only in the field of basic life sciences but also in the field of clinical medicine.…”

“…3B; total histone H3 immunodetected by phosphorylation-independent anti-histone H3 antibody). In addition, phosphorylated amino acid residues present in the three bands were identified by using site-specific anti-histone H3 antibodies against Thr-3 (pT 3 ), Ser-10 (pS 10 ), Thr-11 (pT 11 ), and Ser-28 (pS 28 ).…”