Introducing Cysteines into Nanobodies for Site-Specific Labeling

Simon Boje Hansen, Kasper Røjkjær Andersen*

*Corresponding author for this work

Research output: Contribution to book/anthology/report/proceedingBook chapterResearchpeer-review


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.

Original languageEnglish
Title of host publicationSingle-Domain Antibodies : Methods and Protocols
EditorsGreg Hussack, Kevin A. Henry
Number of pages17
Place of publicationNew York
PublisherHumana Press
Publication date2022
ISBN (Print)978-1-0716-2074-8
Publication statusPublished - 2022
SeriesMethods in Molecular Biology


  • Cysteine
  • Experimental phasing
  • Imaging
  • Maleimide
  • Mercury
  • Nanobody
  • Site-specific labeling


Dive into the research topics of 'Introducing Cysteines into Nanobodies for Site-Specific Labeling'. Together they form a unique fingerprint.

Cite this