Magnetic his dynabeads

Magnetic HIS dynabeads (Thermo Fisher Scientific) were used to deplete mouse sera of antibodies with specificity to MERS S domains, S-2P, S1, S2, and RBD, according to the manufacturer’s protocol. For this, 5 µL sera was added to molar equivalents of depleting protein (calculated from 50 µg/mL foldon) in 445 µL of PBS and incubated for 1 h at room temperature. Magnetic beads were washed three times in PBS, resuspended, and 50 µL was added to each serum-protein mixture and incubated at room temperature for 30 min. Beads were separated from the solution by magnetic strip, and after 5 min, the supernatant was collected and used for future assays.

His-tagged Rab proteins were expressed in Escherichia coli BL21 (DE3) (Agilent Technologies) after induction with 0.5 mM IPTG for 4 h at 37°C. The bacterial cultures were centrifuged and the pellets were resuspended in buffer containing 64 mM Tris-HCl pH 8.5, 8mM MgCl₂, 20 mM β-mercaptoethanol, 0.30 mM PMSF, 0.8 ng lysozyme/gram pellet and 10 μg/mL DNase. The resuspended bacterial cultures were lysed by French press and centrifuged at 48,000 × g for 1 h at 4°C. The soluble fractions containing the expressed His-tagged Rab proteins were purified by using Nickel-nitrilotriacetic acid (Ni-NTA) columns. Amicon® Ultra-15 filtration tubes were used to concentrate the purified proteins and the buffer exchanged with PBS 1×. Pulldown experiments were performed by using magnetic His-dynabeads® (Thermo Fisher). 12 μg of purified His-tagged proteins were bound to Dynabeads and incubated with 200 μL of precleared U2OS cell lysates for 30 min at 4°C. To activate Rab GTPases, purified His-tagged Rabs bound to Dynabeads were loaded with 0.1 mM GTPγS. The beads were washed ten times with buffer containing 3.25 mM Na-P pH 7.4, 79 mM NaCl, 0.01% Tween 20. Bound proteins were eluted with elution buffer (50 mM Na-P pH 8.0, 300 mM NaCl, 0.01% Tween 20, 300 mM Imidazole). Samples were analyzed by using SDS-PAGE and immunoblotting.