Bioorthogonal click chemistry-based labelling of proteins in living neuronal cell lines and primary neurons

Developments in microscopy techniques have brought about a demand for new protein labelling methods. Labelling tags should be small and ideally compatible with live-cell imaging. An elegant protein labelling technique that fulfils these criteria is based on genetic code expansion and bioorthogonal click chemistry. Genetic code expansion allows incorporation of clickable UAAs into proteins of interest which are subsequently directly labelled with small fluorescent dyes using click chemistry. While previous work has mainly focused on easy-to-transfect standard cell lines, recent publications have described applications of this technology in primary neurons. Here, we report a detailed protocol for transfections and bioorthogonal click chemistry-based labelling of intracellular proteins in living primary neurons.