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Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants

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 o...

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Autores principales: Bekešová, Slávka, Komis, George, Křenek, Pavel, Vyplelová, Petra, Ovečka, Miroslav, Luptovčiak, Ivan, Illés, Peter, Kuchařová, Anna, Šamaj, Jozef
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4429547/
https://www.ncbi.nlm.nih.gov/pubmed/26029234
http://dx.doi.org/10.3389/fpls.2015.00336
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author Bekešová, Slávka
Komis, George
Křenek, Pavel
Vyplelová, Petra
Ovečka, Miroslav
Luptovčiak, Ivan
Illés, Peter
Kuchařová, Anna
Šamaj, Jozef
author_facet Bekešová, Slávka
Komis, George
Křenek, Pavel
Vyplelová, Petra
Ovečka, Miroslav
Luptovčiak, Ivan
Illés, Peter
Kuchařová, Anna
Šamaj, Jozef
author_sort Bekešová, Slávka
collection PubMed
description 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.
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spelling pubmed-44295472015-05-29 Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants Bekešová, Slávka Komis, George Křenek, Pavel Vyplelová, Petra Ovečka, Miroslav Luptovčiak, Ivan Illés, Peter Kuchařová, Anna Šamaj, Jozef Front Plant Sci Plant Science 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. Frontiers Media S.A. 2015-05-13 /pmc/articles/PMC4429547/ /pubmed/26029234 http://dx.doi.org/10.3389/fpls.2015.00336 Text en Copyright © 2015 Bekešová, Komis, Křenek, Vyplelová, Ovečka, Luptovčiak, Illés, Kuchařová and Šamaj. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Bekešová, Slávka
Komis, George
Křenek, Pavel
Vyplelová, Petra
Ovečka, Miroslav
Luptovčiak, Ivan
Illés, Peter
Kuchařová, Anna
Šamaj, Jozef
Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants
title Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants
title_full Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants
title_fullStr Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants
title_full_unstemmed Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants
title_short Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants
title_sort monitoring protein phosphorylation by acrylamide pendant phos-tag™ in various plants
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4429547/
https://www.ncbi.nlm.nih.gov/pubmed/26029234
http://dx.doi.org/10.3389/fpls.2015.00336
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