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Immobilized enzyme technologies in protein phosphorylation and its analysis

Authors: Slováková Marcela | Hromádková Lenka | Fojtíková Markéta | Strouhalová Eliška | Kupčík Rudolf | Vajrychová Marie | Bílková Zuzana

Year: 2020

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	Immobilized enzyme technologies in protein phosphorylation and its analysis	Protein phosphorylation, as a major post-translational modification, has a major influence on the physiological functions of proteins, especially those that are in native conformation and are involved in many neurodegenerative diseases. However, structural and functional studies of such proteins require fully defined phosphorylation, including non-physiological ones. In this presentation, application of magnetic immobilized serine, threonine and tyrosine protein kinases will be presented. The principle of magnetic separation of particles with immobilized kinases facilitates and increases the laboratory comfort of the necessary separation of phosphorylated substrates. Immobilized enzyme activity and stability was verified using peptide substrates and evaluated by mass spectrometry analysis, western blot with phosphospecific antibodies and pIMAGOTM dendrimer. Immobilized kinases have utility in sample preparation and are also part of specialized biosensors. Compared to soluble kinases, immobilized kinases have the advantages of, in particular, reuse, long-term storage and higher enzyme stability. Thus, when phosphorylating proteins and peptides with immobilized protein kinases, the resulting product is highly pure and not contaminated with soluble enzymes. If multiple substrate phosphorylations are desired, carriers with immobilized kinases or immobilized kinases in a mixture can be sequentially used [1]. Methods used to immobilize kinases to solid supports include adsorption [2], covalent bonds [1,3], and glutaraldehyde capture [4].	Immobilized; enzyme; protein phosphorylation;