Figure 1.

Engineering of Quad molecules. (A) Schematic structural overview of the different Quad formats generated using the p53 tetramerization domain. (B) Nonreducing and reducing colloidal blue-stained SDS-PAGE analysis of the eight Quad constructs and the nanobody. (C) Assessment of purity and monomeric assembly of the Quads via size-exclusion chromatography analysis displayed according to their binding domain valency. The chromatograms were obtained on a HiLoad Superdex200 Increase 10/300 GL column with PBS as an eluent. (D) Binding curve established with FIDA showing the apparent hydrodynamic radius of the indicator α-cbtx-Alexa488 (100 nM) as a function of anti-α-cbtx (0–2.1 μM) in PBST buffer. The K_D values were calculated from the binding isotherm and are available in Table S2. Represented results are from a single experiment with technical repeats performed in duplicate. *Denotes Quad formats that had increased interaction with the FIDA capillary. (E) ELISA binding assay of Quads to immobilized α-cbtx. Each data point represents the mean of two independent experiments ± SD. The K_D values were calculated from the binding curves and are available in Table S4.