Selection and characterization of UbV‐phage binding to yUIM‐1

A peptide containing the first UIM of Vps27 (GGGGAADEEELIRKAIELSLKESRNSGGY) was biotinylated with N‐hydroxysuccinimidyl d‐biotin‐15‐amido‐4, 7, 10, 13‐tetraoxapentadecylate (NHS‐PEO₄‐Biotin) in accordance with the manufacturer's instructions (Thermo Fisher Scientific). The biotinylated peptide was immobilized in 96‐well Nunc‐Immuno MAXISORP plates (Thermo Scientific) coated with streptavidin (New England Biolabs), and phage pools representing UbV library 231 were cycled through five rounds of binding selections with the immobilized peptide, as described.36, 37 UbV‐phage clones that bound to the peptide but not to streptavidin or BSA were identified by clonal phage ELISAs and were subjected to DNA sequence analysis to decode the sequences of the displayed UbVs, as described.31