<p>(<b>A</b>) Binding of recombinant C11L34 antibody to a single or tandem GCN epitope placed within the PCDH15 sequence and expressed in RPMI 2650 cells. Binding was normalized to the signal of an anti-PCDH15 antibody. The binding to the tandem epitope was about 15x better, if the linker was 19 amino acids (green line) but not if the linker was shorter (magenta). (<b>B</b>) Immunoblots of cell extracts showing much stronger binding to the tandem epitope with longer linker. Lysates from HEK cells expressing PCDH15 with different tags were run on two identical gels; one was probed with the divalent C11L34 anti-GCN at a fixed concentration and one with anti-PCDH15 as a loading control. Full sized immunoblots are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150125#pone.0150125.s003" target="_blank">S3 Fig</a>. (<b>C</b>) Binding of recombinant anti-THAP antibody or biotinylated BTX to a single or tandem HAP epitope placed within the PCDH15 sequence, normalized to the anti-PCDH15 signal. Anti-THAP avidity for the THAP tag was about 7X better than to the single HAP tag. (<b>D</b>) Immunoblots of cell extracts showing much stronger binding to the tandem epitope than to the single HAP. Protocol as in (B). Lower panel shows the binding to the tagged PCDH15 by anti-THAP relative to anti-PCDH15. Full sized immunoblots are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150125#pone.0150125.s004" target="_blank">S4 Fig</a>. (<b>E</b>) Biacore surface plasmon resonance analysis of binding between the recombinant antibodies to HAP or THAP peptides. One antibody (anti-THAP; shown as BTX:BTX) contained symmetric BTX binding domains, while another (BTX:con) was asymmetric with one arm containing BTX and the other an irrelevant control binding domain. The THAP peptide showed higher affinity, but it was substantially higher only for the anti-THAP antibody, and only when the linker length was 10 rather than 14 amino acids. Representative traces are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150125#pone.0150125.s001" target="_blank">S1 Fig</a>. Measured rate constants and dissociation constants are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150125#pone.0150125.s005" target="_blank">S1 Table</a>. (<b>F</b>) Single-molecule pull-down assay. Cell lysates containing monomeric YFP tagged with THAP and 6xHis were applied at different dilutions to slides with immobilized anti-His or anti-THAP antibodies, or anti-HA as a negative control. Insets show molecules pulled down. Full immunoblots for panels B and D are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150125#pone.0150125.s003" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150125#pone.0150125.s004" target="_blank">S4</a> Figs.</p
