Three methods for examining the de novo proteome of microglia using BONCAT bioorthogonal labeling and FUNCAT click chemistry.

Bioorthogonal labeling and click chemistry techniques allow the detailed examination of cellular physiology through tagging and visualizing newly synthesized proteins. Here, we describe three methods applying bioorthogonal non-canonical amino acid tagging and fluorescent non-canonical amino acid tagging to quantify protein synthesis in microglia. We describe steps for cell seeding and labeling. We then detail microscopy, flow cytometry, and Western blotting techniques. These methods can be easily adapted for other cell types to explore cellular physiology in health and disease. For complete details on the use and execution of this protocol, please refer to Evans et al. (2021)..