Chapter title |
Affinity-Based SDS PAGE Identification of Phosphorylated Arabidopsis MAPKs and Substrates by Acrylamide Pendant Phos-Tag™.
|
---|---|
Chapter number | 5 |
Book title |
Plant MAP Kinases
|
Published in |
Methods in molecular biology, May 2014
|
DOI | 10.1007/978-1-4939-0922-3_5 |
Pubmed ID | |
Book ISBNs |
978-1-4939-0921-6, 978-1-4939-0922-3
|
Authors |
Komis G, Takáč T, Bekešová S, Vadovič P, Samaj J, George Komis, Tomáš Takáč, Slávka Bekešová, Pavol Vadovič, Jozef Šamaj |
Editors |
George Komis, Jozef Šamaj |
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. |
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