Protocol to generate fast-dissociating recombinant antibody fragments for multiplexed super-resolution microscopy.

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,

SUBMITTER: Zhang Q 

PROVIDER: S-EPMC10468357 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature