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. 2026 Jan 24;37(2):281–292. doi: 10.1021/acs.bioconjchem.5c00519

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© 2026 The Authors. Published by American Chemical Society

This article is licensed under CC-BY 4.0

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NanoBondy covalent conjugation to recombinant CD45. (A) AlphaFold prediction of 2H5 nanobody (purple) interaction with CD45d1d2 (green). Magenta represents the site for a disulfide clamp, with lysines on CD45d1d2 colored pink and the terminal aspartate in cyan. (B) MBP fusion improved the CD45 gel-based analysis. CD45d consists of MBP fused to domains 1 and 2 of CD45. PNGase F digestion decreased heterogeneous mobility of CD45d1d2 and CD45d upon SDS-PAGE with Coomassie staining. (C) Individual protein components for the conjugation assay in (D). CD45d, sfGFP, ODC, and PNGase F were validated by SDS-PAGE/Coomassie staining. (D) Specificity of the NanoBondy reaction with recombinant CD45. Anti-CD45 2H5 R72C or anti-IgG NanoBondy-SPM was incubated with CD45d, each at 10.5 μM, for 1 h at 37 °C in HBS ± calcium, followed by SDS-PAGE with Coomassie staining. ODC, sfGFP, and anti-IgG NanoBondy were used as negative controls for reaction specificity. Hydroxylamine was used as a competing nucleophile to block reactivity. Colon represents a covalent conjugate. The experiment was conducted once.