Supplementary Figure 4: Discovery of nanobodies with nonpurified antigen
From: Yeast surface display platform for rapid discovery of conformationally selective nanobodies
(a) Conditioned medium containing adiponectin (left lane) was used for selection of nanobodies. It shows a complex mixture of proteins as assessed by SDS-PAGE. For reference, purified adiponectin is shown in the right lane. Adiponectin exists as a mix of 16-mers, hexamers, and trimers. (b) Schematic of selection process. Fluorescent anti-FLAG antibody was used to specifically mark those yeast cells that display adiponectin-binding nanobodies. (c) Flow cytometry analysis of final clone pool, showing that the library was highly enriched in adiponectin-binding clones. (d) Sequences of five selected clones showed highly diverse CDR3 sequence composition and length. (e) Binding assessed using in vitro pull-down with purified adiponectin globular domain. (f) Binding to adiponectin was further confirmed in vitro using surface plasmon resonance. Kinetic fit is shown for clone Nb.AQ103.
