Imami Koshi, Yasuda Tomoharu
Department of Molecular and Cellular BioAnalysis, Kyoto University, Kyoto, Japan.
Department of Molecular and Cellular Biology, Kyushu University, Fukuoka, Japan.
Bio Protoc. 2019 Apr 20;9(8):e3215. doi: 10.21769/BioProtoc.3215.
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.
蛋白质合成是维持细胞蛋白质稳态最基本的生物学过程之一。叠氮高丙氨酸（AHA）是甲硫氨酸的一种非放射性且可“点击”的氨基酸类似物，它能够掺入新合成的蛋白质中。因此，通过“点击”化学进行荧光标记，可以检测和定量AHA标记的新生蛋白质。在此，我们描述一种通过AHA标记和流式细胞术来测量蛋白质合成的方案。利用对不同细胞群体进行门控的方法，我们提供了一个基于流式细胞术分析细胞周期中蛋白质合成的典型示例。虽然在本方案中我们使用的是小鼠B细胞，但该方法可轻易应用于任何细胞类型和生物体。
