title: Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants.
abstract: The aim of the present study is to rationalize acrylamide pendant Phos-Tag™ in-gel discrimination of phosphorylated and non-phosphorylated plant protein species with standard immunoblot analysis, and optimize sample preparation, efficient electrophoretic separation and transfer. We tested variants of the method including extraction buffers suitable for preservation of phosphorylated protein species in crude extracts from plants and we addressed the importance of the cation (Mn(2+) or Zn(2+)) used in the gel recipe for efficient transfer to PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of Medicago sativa stress-induced mitogen activated protein kinase (SIMK) in stress-treated wild type plants and transgenic SIMKK RNAi line. We further show the hyperosmotically-induced phosphorylation of the previously uncharacterized HvMPK4 of barley. The method is validated using inducible phosphorylation of barley and wheat α-tubulin and of Arabidopsis MPK6. Acrylamide pendant Phos-Tag™offers a flexible tool for studying protein phosphorylation in crops and Arabidopsis circumventing radioactive labeling and the use of phosphorylation specific antibodies.
authors.affiliation: Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University Olomouc Olomouc, Czech Republic.
authors.forename: Slávka
authors.initials: S
authors.lastname: Bekešová
authors.affiliation: Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University Olomouc Olomouc, Czech Republic.
authors.forename: George
authors.initials: G
authors.lastname: Komis
authors.affiliation: Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University Olomouc Olomouc, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Křenek
authors.affiliation: Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University Olomouc Olomouc, Czech Republic.
authors.forename: Petra
authors.initials: P
authors.lastname: Vyplelová
authors.affiliation: Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University Olomouc Olomouc, Czech Republic.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Ovečka
authors.affiliation: Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University Olomouc Olomouc, Czech Republic.
authors.forename: Ivan
authors.initials: I
authors.lastname: Luptovčiak
authors.affiliation: Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University Olomouc Olomouc, Czech Republic.
authors.forename: Peter
authors.initials: P
authors.lastname: Illés
authors.affiliation: Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University Olomouc Olomouc, Czech Republic.
authors.forename: Anna
authors.initials: A
authors.lastname: Kuchařová
authors.affiliation: Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University Olomouc Olomouc, Czech Republic.
authors.forename: Jozef
authors.initials: J
authors.lastname: Šamaj
keywords: Arabidopsis thaliana; Hordeum vulgare; Medicago sativa; SDS-PAGE Phos-TagTM; Triticum turgidum; mitogen activated protein kinase; protein phosphorylation