Figure 5. AHA labeling of newly synthesized proteins. (A) Structure of L-azidohomoalanine (AHA), an analog of methionine containing an azide moiety. (B) Click azide/alkyne reaction. The azide and alkyne moieties are interchangeable, where the molecule can be labeled with an alkyne and react with a fluorophore- or hapten-azide. (C) Workflow for AHA labeling-based quantitative analysis of protein degradation. The bio-orthogonal noncanonical amino acid tagging (BONCAT) strategy for labeling, detection and identification of newly synthesized proteins. Cells are incubated with AHA to allow protein synthesis. After incubation, cells are harvested, fixed, and permeabilized. Nascent protein synthesis is detected following a click reaction with Rhodamine B alkyne and analysis is performed using flow cytometry.