Measuring Protein Synthesis during Cell Cycle by Azidohomoalanine (AHA) Labeling and Flow Cytometric Analysis

Protein synthesis is one of the most fundamental biological processes to maintain cellular proteostasis. Azidohomoalaine (AHA) is a non-radioactive and "clickable" amino acid analog of methionine which can be incorporated into newly synthesized proteins. Thus, AHA-labeled nascent proteins can be detected and quantified through fluorescent labeling by "click" chemistry. Here we describe a protocol to measure protein synthesis by AHA labeling and flow cytometry. Taking advantage of gating different cell populations, we provide a typical example of the flow cytometric-based analysis of protein synthesis during the cell cycle. While we used mouse B cells in this protocol this method can be readily applied to any cell types and organisms.