Wang Yu, Shen Zhixun, Wan Zhengpeng, Liu Wanli
Ministry of Education Key Laboratory of Protein Sciences, Center for Life Sciences, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Institute for Immunology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
Department of Biological Engineering, Massachusetts Institutes of Technology, Cambridge, MA, USA.
Bio Protoc. 2020 Sep 20;10(18):e3767. doi: 10.21769/BioProtoc.3767.
B lymphocyte activation is regulated by its membrane-bound B cell receptors (BCRs) upon recognizing diverse antigens. It is hypothesized that antigen binding would trigger conformational changes within BCRs, followed by a series of downstream signaling activation. To measure the BCR conformational changes in live cells, a fluorescent site-specific labeling technique is preferred. Genetically encoded fluorescent tags visualize the location of the target proteins. However, these fluorescent proteins are large (~30 kDa) and would potentially perturb the conformation of BCRs. Here, we describe the general procedures of utilizing short tag-based site-specific labeling methodologies combining with fluorescence resonance energy transfer (FRET) assay to monitor the conformational changes within BCR extracellular domains upon antigen engagement.
B淋巴细胞激活是由其膜结合的B细胞受体（BCR）识别多种抗原后进行调节的。据推测，抗原结合会触发BCR内的构象变化，随后是一系列下游信号激活。为了测量活细胞中BCR的构象变化，荧光位点特异性标记技术是首选。基因编码的荧光标签可可视化目标蛋白的位置。然而，这些荧光蛋白很大（约30 kDa），可能会干扰BCR的构象。在这里，我们描述了利用基于短标签的位点特异性标记方法结合荧光共振能量转移（FRET）测定来监测抗原结合后BCR细胞外结构域内构象变化的一般程序。
