Preparation of Protein Lysates Using Biorthogonal Chemical Reporters for Click Reaction and in-Gel Fluorescence Analysis.

Bioorthogonal chemical reporters are non-native chemical handles introduced into biomolecules of living systems, typically through metabolic or protein engineering. These functionalities can undergo bioorthogonal reactions, such as copper-catalyzed alkyne-azide cycloaddition (CuAAC), with small-molecule probes, enabling the tagging and visualization of biomolecules. This approach has greatly enhanced our understanding of cellular dynamics, enzyme targeting, and protein post-translational modifications. Herein, we report a protocol for preparing protein lysates for click reaction and in-gel fluorescence analysis, exemplified using alk-16, a terminal alkyne-functionalized stearic acid analog, to investigate proteins with fatty acylation. This protocol provides methods for the fluorescent visualization of chemical reporter-labeled proteomes or individual proteins of interest (POIs). Key features • Metabolic incorporation of bioorthogonal chemical reporters into proteins in living cells. • Visualization of proteomes or specific proteins labeled with chemical reporters via in-gel fluorescence analysis. • Reliable, non-radioactive methods for investigating protein fatty acylation and other post-translational modifications.