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Abstract：
Multiplexed high-density label super-resolution microscopy image reconstruction by integrating exchangeable single-molecule localization (IRIS) enables elucidating fine structures and molecular distribution in cells and tissues. However, fast-dissociating binders are required for individual targets. Here, we present a protocol for generating antibody-based IRIS probes from existing antibody sequences. We describe steps for retrieving antibody sequences from databases. We then detail the construction, purification, and evaluation of recombinant probes after site-directed mutagenesis at the base of complementarity-determining region loops. The protocol accelerates dissociation rates without compromising the binding specificity. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1.
摘要：
通过整合可交换的单分子定位（IRIS），多路复用的高密度标记超分辨率显微镜图像重建能够阐明细胞和组织中的精细结构和分子分布。然而,单个靶标需要快速解离的结合剂。这里,我们提出了从现有抗体序列产生基于抗体的IRIS探针的方案.我们描述了从数据库检索抗体序列的步骤。然后我们详细说明施工，净化,并在互补决定区环的碱基处进行定点诱变后评估重组探针。该方案加速解离速率而不损害结合特异性。有关此协议的使用和执行的完整详细信息，请参阅Zhang等人。（2022）.1。
