Dynabeads his tag isolation and pulldown

Rosetta E. coli cells containing the UME6- pET14b plasmid were grown at 37 °C in 1 L of LB ampicillin medium supplemented with 1% of glucose up to OD_600nm 0.6 expression was induced with 0.15 g/L IPTG at 20 °C for 4 h. Cells were harvested by centrifugation and pellet was resuspended in 20 ml of binding buffer (50 mM sodium phosphate ph7.4, 300 mM NaCl, 1 mM PMSF, 0.01% tween20, lysozyme 0.2 mg/ml, 1 mM MgCl2, 2000u DNase and a tablet of cOmplete™EDTA-free Protease Inhibitor Cocktail), incubated for 30 min on ice and sonicated twice for 4 min, followed by centrifugation at 10.000 × g for 15 min. The supernatant was incubated 10 min with the Dynabeads™ His-Tag Isolation and Pulldown (ThermoFisher). Beads were washed 4 times with wash buffer (50 mM sodium phosphate ph7.4, 300 mM NaCl, 5 mM imidazole, 0.01% tween20). Finally, protein was eluted with the elution buffer (50 mM sodium phosphate ph7.4, 300 mM NaCl, 300 mM imidazole, 0.01% tween20) and dialyzed overnight with 10 mM sodium phosphate pH 7.4, 50 mM NaCl and 10 µM ZnCl₂ buffer. Ume6 protein was visualized by SDS-PAGE and coomassie brilliant blue staining.

Selected nanobodies were coupled to Co²⁺-coated magnetic Dynabeads (Dynabeads^® His-Tag Isolation and Pulldown, Thermo Fisher Scientific) to make them multivalent, using the buffers described in the manual. Nanobodies (24 µg) were added to 300 µg of Dynabeads and the mixture was incubated at room temperature during 10 min on a roller. Hereafter, the beads were washed three times to remove the unbound nanobodies and the nanobody-coupled beads were stored in pull-down buffer at 4 °C.
Campylobacter jejuni KC40 cells were harvested from NB2-agar plates with NB2 medium and a suspension with OD₆₆₀ 2.0 was used for the agglutination assay. The bacterial cells and nanobody-coupled beads were mixed in a 1:4 ratio in a final volume of 10 µL on a slide. The slides were incubated at room temperature and examined visually and by phase contrast microscopy for agglutination. As a negative control, the agglutination of Campylobacter bacteria in the presence of beads coupled with anti-F4 nanobodies was assessed [48 (link), 49 (link)].