title: Affinity-based SDS PAGE identification of phosphorylated Arabidopsis MAPKs and substrates by acrylamide pendant Phos-Tag™.
abstract: Protein phosphorylation is the most abundant and best studied protein posttranslational modification, dedicated to the regulation of protein function and subcellular localization as well as to protein-protein interactions. Identification and quantitation of the dynamic, conditional protein phosphorylation can be achieved by either metabolic labeling of the protein of interest with (32)P-labeled ATP followed by autoradiographic analysis, the use of specific monoclonal or polyclonal antibodies against the phosphorylated protein species and finally by phosphoproteome delineation using mass spectrometry.Hereby we present a fourth alternative which relies on the enforced-affinity-based-electrophoretic separation of phosphorylated from non-phosphorylated protein species by standard SDS-PAGE systems co-polymerized with Phos-Tag™ and Mn(2+) or Zn(2+) cations. Phosphate groups of phosphorylated Ser, Thr, and Tyr residues form complexes with Mn(2+) and Zn(2+) cations with polyacrylamide immobilized Phos-Tag™. Following appropriate treatment of the gels, separated proteins can be quantitatively transferred to PVDF or nitrocellulose membranes and probed with common-not phosphorylation state specific-antibodies and delineate the occurrence of a certain phosphoprotein species against its non-phosphorylated counterpart.
authors.affiliation: Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University Olomouc, Šlechtitelů 11, 783 71, Olomouc, Czech Republic, georgios.komis@upol.cz.
authors.forename: George
authors.initials: G
authors.lastname: Komis
authors.forename: Tomáš
authors.initials: T
authors.lastname: Takáč
authors.forename: Slávka
authors.initials: S
authors.lastname: Bekešová
authors.forename: Pavol
authors.initials: P
authors.lastname: Vadovič
authors.forename: Jozef
authors.initials: J
authors.lastname: Samaj
mesh_terms: D020106:Acrylamide / Q000737:chemistry*; D017360:Arabidopsis / Q000201:enzymology* / Q000254:growth & development; D010582:Bacteriophage lambda / Q000201:enzymology; D046508:Culture Techniques; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008567:Membranes, Artificial; D020928:Mitogen-Activated Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism*; D019800:Phenol / Q000737:chemistry; D010750:Phosphoproteins / Q000302:isolation & purification* / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011145:Polyvinyls / Q000737:chemistry; D005465:Fluorocarbon Polymers
chemical_list: D020106:Acrylamide; D008567:Membranes, Artificial; D020928:Mitogen-Activated Protein Kinases; D019800:Phenol; D010750:Phosphoproteins; D010744:Phosphoric Monoester Hydrolases; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers