Dynabeads m 270 beads

Chemical conjugation of the anti-GFP nanobody to epoxyl Dynabeads^® M-270 beads (Invitrogen, Thermo Fisher Scientific) was performed as previously described^{22 (link)}. Briefly, 10 mg of epoxyl Dynabeads^® M-270 beads were conjugated with 100 µg Lag16-2K/R anti-GFP nanobody. Beads were washed for 15 min with 0.1 M sodium phosphate buffer pH 7.4 and for 5 min with 0.1 M sodium phosphate buffer pH 8 rocking. Antibody mix was prepared by adding 100 µg nanobody, 0.1 M sodium phosphate buffer pH 8–200 µl final volume and 1 M ammonium sulfate in 0.1 M sodium sulfate pH 7.4 (added dropwise). Antibody mix was centrifuged at maximum speed on a bench top centrifuge for 5 min before being added to the washed beads. Antibody mix and beads were incubated ~20 h at 30 °C tilted-rotating in a round bottom 2 ml microcentrifuge tube. After incubation conjugated beads were washed with 100 mM glycine pH 2.5, 10 mM Tris-HCl pH 8.8 and 100 mM triethylamine freshly prepared to quench the reaction, wash away unbound nanobody and block unreacted epoxyl groups on the beads. Finally, anti-GFP conjugated beads were washed with PBS for 5 min 4 times, with 0.5% NP40 in PBS one time for 5 min and a second time for 15 min rocking, and with PBS for 5 min before storage at −20 °C in 30% glycerol and 0.02% sodium azide in PBS until further use.