Antigen Identification for Cell-Binding Antibodies Using Ligand-Directed Crosslinking and Biotin Transfer

  • Tammy-Lynn Tremblay
  • Jennifer J. HillEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2024)


Panning approaches using antibody libraries often result in the isolation of antibodies that bind to cells through an unknown cellular receptor. Here, we describe a protocol that uses ligand-directed crosslinking with the aminooxy-sulfhydryl-biotin (ASB) trifunctional crosslinker followed by a proteomic analysis to identify the cellular receptors for orphan ligands. We describe the synthesis of the ASB crosslinker, labelling of the ligand with ASB, and cell binding of the labelled ligands. Next, biotin affinity purification and trypsin digestion of cell surface proteins that have been crosslinked by ASB are described. Lastly, several hints and tips to improve the proteomic analysis for these types of samples are provided.

Key words

Receptor identification Crosslinking Proteomics Mass spectrometry Antibodies 



We would like to thank the National Research Council Canada for supporting this research.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Human Health TherapeuticsNational Research Council CanadaOttawaCanada

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