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

Tammy-Lynn Tremblay1, Jennifer J Hill2

  • 1Human Health Therapeutics, National Research Council Canada, Ottawa, ON, Canada.

Methods in Molecular Biology (Clifton, N.J.)
|August 1, 2019
PubMed

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.

Keywords:
AntibodiesCrosslinkingMass spectrometryProteomicsReceptor identification

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