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Immobilized ERK2 and GSK-3beta magnetic microparticles for targeted protein phosphorylation
Authors: Slováková Marcela | Hromádková Lenka | Kupčík Rudolf | Řípová Daniela | Bílková Zuzana
Year: 2016
Type of publication: ostatní - přednáška nebo poster
Page from-to: nestránkováno
Titles:
Language Name Abstract Keywords
eng Immobilized ERK2 and GSK-3beta magnetic microparticles for targeted protein phosphorylation Introduction: Micro and nanoparticles with immobilized enzymes as recoverable, stable and specific catalysts are applied in a variety of technologies, in biomedical applications and widely in research. Magnetic microparticles bring the advantage of the non-contaminating and very specific and sensitive reaction on their substrates, peptides and proteins. This work is based on the use of proline-directed protein kinases: extracellular signal-regulated kinase (ERK2) and glycogen synthase kinase 3β (GSK-3β) immobilized to magnetic microparticles for targeted recombinant tau 1-441 phosphorylation. Methods: In order to obtain highly efficient carrier for the targeted phosphorylation of peptides/proteins, two kinases ERK2 and GSK-3 were immobilized to various superparamagnetic beads with carboxylic, aldehyde, and metal cations Ni2+ or Co3+ functionalities. Relevant methods of covalent immobilization, non-oriented and oriented, were chosen and reaction conditions were optimized. Phosphorylation of low molecular peptides and operational and storage stabilities of kinase-superparamagnetic particles were performed. Soluble and immobilized ERK2 and GSK-3β were applied for recombinant tau 1-441 phosphorylation. Tryptic phosphopeptides enrichment was performed by ion-metal affinity chromatography using TiO2 magnetic nanoparticles. The level and the position of phosphorylation sites were identified by using MALDI-LTQ-Orbitrap MS. Western blot was carried out to confirm phosphorylation of tau 1–441 (ENZO and rPeptide) by soluble or immobilized ERK2 and GSK-3β kinases using specific anti-tau and anti-phospho-tau antibodies. Acknowledgement: EU project NADINE No. 246513 Immobilized kinase; ERK2; GSK-3beta; magnetic microparticles; targeted phosphorylation

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