Nanobody Expression and Purification

Miro1-Nbs were expressed and purified as previously described (Maier et al., 2015 (link); Wagner and Rothbauer, 2021 (link)). Bivalent Nbs were expressed using the ExpiCHO™ system (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s protocol. For quality control, all purified proteins were analyzed by SDS-PAGE according to standard procedures. Protein samples were denatured (5 min, 95°C) in 2x SDS-sample buffer containing 60 mM Tris/HCl, pH 6.8; 2% (w/v) SDS; 5% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, 0.02% bromphenol blue prior to analysis. All proteins were visualized by InstantBlue Coomassie (Expedeon) staining. For immunoblotting, proteins were transferred to nitrocellulose membrane (GE Healthcare) and detection was performed using anti-His primary antibody (Penta-His Antibody, #34660, Qiagen) followed by donkey-anti-mouse secondary antibody labelled with AlexaFluor647 (Invitrogen). A Typhoon Trio scanner (GE-Healthcare, excitation 633 nm, emission filter settings 670 nm BP 30) was used for the readout of fluorescence signals.