biostudies-literatureRuggiero EFondazione AIRC per la ricerca sul cancro ETSSlovenian Research and Innovation Agencygkae1305https://www.ebi.ac.uk/biostudies/studies/S-EPMC11734697biostudies-literatureUnknown53(2)i-Motifs (iMs) are quadruplex nucleic acid conformations that form in cytosine-rich regions. Because of their acidic pH dependence, iMs were thought to form only in vitro. The recent development of an iM-selective antibody, iMab, has allowed iM detection in cells, which revealed their presence at gene promoters and their cell cycle dependence. However, recent evidence emerged which appeared to suggest that iMab recognizes C-rich sequences regardless of their iM conformation. To further investigate the selectivity of iMab, we examined the binding of iMab to C-rich sequences, using a combination of pull-down and western blot assays. Here, we observe that the composition of buffers used during binding and washing steps strongly influences the selectivity of antibody binding. In addition, we dNucleic acids researchThe iMab antibody selectively binds to intramolecular and intermolecular i-motif structures.PMC1173469721850P1-0242Zanin IRuggiero EMarusic MPlavec JChrist DPena Martinez CDRichter SNfalseThe iMab antibody selectively binds to intramolecular and intermolecular i-motif structures.i-Motifs (iMs) are quadruplex nucleic acid conformations that form in cytosine-rich regions. Because of their acidic pH dependence, iMs were thought to form only in vitro. The recent development of an iM-selective antibody, iMab, has allowed iM detection in cells, which revealed their presence at gene promoters and their cell cycle dependence. However, recent evidence emerged which appeared to suggest that iMab recognizes C-rich sequences regardless of their iM conformation. To further investigate the selectivity of iMab, we examined the binding of iMab to C-rich sequences, using a combination of pull-down and western blot assays. Here, we observe that the composition of buffers used during binding and washing steps strongly influences the selectivity of antibody binding. In addition, we d2025-01-01T00:00:00Z2025 Jan2025-04-04T08:51:02.946Z2025-04-04T08:51:02.946ZS-EPMC117346973981751110.1093/nar/gkae1305