Detekce metabolicky značených proteinů a RNA pomocí Click reakce na membráně [Click-on-Membrane for Detection of Metabolically Labelled Proteins and RNA]
Klíčová slova:
Click chemie, BONCAT, bioortogonální nekanonické značení aminokyselin, 2D elektroforéza, elektroblotAbstrakt
Bioorthogonal chemistry provides one of the possibilities to modify various biomolecules in their native environment. The combination of Click chemistry with the BONCAT method (bioorthogonal non-canonical amino acid tagging) is widely used for tagging and analysis of newly synthesized proteins, which are clearly distinguishable from the pre-existing protein pool. However, the commonly used procedure results in low quality 2D electrophoretic profiles. We put a lot of effort into obtaining clear results using a standard Click protocol, with a negligible effect. Here we describe a Click-on-membrane approach which we successfully used not only to monitor de novo protein synthesis but also to detect newly synthesized RNA.
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