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Immobilisation of tyrosine protein kinase Src to magnetic beads for in vitro phosphorylation of proteins
Authors: Bednářová Šárka | Morávek Ondřej | Kupčík Rudolf | Fojtíková Markéta | Slováková Marcela
Year: 2024
Type of publication: ostatní - přednáška nebo poster
Page from-to: nestránkováno
Titles:
Language Name Abstract Keywords
eng Immobilisation of tyrosine protein kinase Src to magnetic beads for in vitro phosphorylation of proteins For protein phosphorylation, it is possible to use both immobilised protein kinases and free ones. However, immobilised kinases have great advantages. It is possible to stop the reaction immediately, isolate protein kinases easily from the mixture, and obtain a pure product, for instance, model proteins and peptides; enzymes could be used repeatedly. For the best kinase activity, it is necessary to choose the ideal immobilisation method and the type of beads. In our experiments, Src kinase was conjugated to superparamagnetic beads with micrometre diameters, orientated and random immobilisation was tested, and the effect of the present BSA. Subsequently, the protein kinase immobilised on the beads was used to phosphorylate the peptide substrate, followed by analysis with MALDI LTQ Orbitrap XL MS, and the degree of phosphorylation was calculated from the obtained spectra. Non-oriented conjugation was formed using the carbodiimide method; covalent linkage between the carboxylic group of beads and the amino group of Src kinase. We used several available superparamagnetic beads: Sera Mag speed beads, SiMAG, Dynabeads, and ProMag. The highest kinase activity we obtained with the Sera Mag speed beads was only 38.8 % after 48 hours of phosphorylation. After we modified the conjugation reaction with 0.05% BSA [1], the activity of Src kinase increased to 93.2 % after 48 hours of peptide phosphorylation. Additionally, we also tested the storage activity. Kinase activity decreased only by 10.82 % in 4 weeks of storage. In previous experiments without the presence of BSA, the decrease during 4 weeks of storage was greater, by 33.61 %. This immobilised kinase was used to phosphorylate model proteins related to neurodegenerative diseases, tau protein, alpha-synuclein, and myelin basic protein. Protein phosphorylation detection was performed using Western blot immunodetection using specific antiphosphotyrosine antibodies and MALDI LTQ Orbitrap XL MS. Immobilisation; tyrosine protein kinase; Src; magnetic beads; phosphorylation; myelin basic protein

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