Bastide Amandine, Yewdell Jonathan W, David Alexandre
IGF, CNRS, INSERM, Univ. Montpellier, F-34094 Montpellier, France.
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, USA.
Bio Protoc. 2018 Jan 5;8(1). doi: 10.21769/BioProtoc.2669.
While isotopic labeling of amino acids remains the reference method in the field for quantifying translation rate, it does not provide any information on spatial localization of translation sites. The rationale behind developing the ribopuromycylation method (RPM) was primarily to map translation sites at the sub-cellular level while avoiding detection of newly synthesized proteins released from ribosomes. RPM visualizes actively translating ribosomes in cells via standard immunofluorescence microscopy in fixed and permeabilized cells using a puromycin-specific monoclonal antibody to detect puromycylated nascent chains trapped on ribosomes treated with a chain elongation inhibitor.
虽然氨基酸的同位素标记仍然是该领域量化翻译速率的参考方法，但它无法提供关于翻译位点空间定位的任何信息。开发核糖体嘌呤霉素化方法（RPM）背后的基本原理主要是在亚细胞水平上绘制翻译位点，同时避免检测从核糖体释放的新合成蛋白质。RPM通过标准免疫荧光显微镜在固定和通透的细胞中可视化细胞中正在进行翻译的核糖体，使用嘌呤霉素特异性单克隆抗体检测被困在用链延伸抑制剂处理的核糖体上的嘌呤霉素化新生链。
