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Introducing Cysteines into Nanobodies for Site-Specific Labeling

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We have developed a generally applicable methodology for cysteine mutagenesis of nanobody (Nb) framework region serine residues. This strategy allows for subsequent labeling with thiol-reactive compounds without disrupting Nb antigen binding. We provide a protocol for production, labeling, and affinity determination of cysteine-engineered Nbs (cys-Nbs) with Alexa Fluor 488-maleimide and the mercury compound para-chloromercuribenzoic acid (PCMB). Alexa Fluor 488- and PCMB-labeled cys-Nbs can be used for immunofluorescence microscopy and experimental phasing in crystallography, respectively.

TitelSingle-Domain Antibodies : Methods and Protocols
RedaktørerGreg Hussack, Kevin A. Henry
Antal sider17
UdgivelsesstedNew York
ForlagHumana Press
ISBN (trykt)978-1-0716-2074-8
StatusUdgivet - 2022
SerietitelMethods in Molecular Biology

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