Dynabeads his tag magnetic beads

Direct binding between Rab5B and FHIP1B was assessed by incubating 15 μM 6xHis-SNAP-Rab5B with 25-molar excess of GMP-PNP (Abcam) or GPD (Abcam) in nucleotide buffer (50 mM Tris–HCl, pH 7.5, 300 mM NaCl, 10 mM EDTA) overnight at 4°C. The next day, reactions were supplemented with 15 mM MgCl₂ and nucleotide-bound 6xHis-SNAP-Rab5B was diluted to 5 μM in Ni-binding buffer (50 mM Tris–HCl, pH 7.5, 500 mM NaCl, 20 mM imidazole, 0.1% casein). 6xHis-SNAP-Rab5B was then captured onto 20 μl Dynabeads His-Tag Magnetic Beads (Thermo Fisher Scientific) and washed three times in Ni-binding buffer in 2 ml Protein Lo Bind Tubes (Eppendorf). Bead conjugated 6xHis-SNAP-Rab5B was incubated for 15 min in room temperature with gentle agitation. The beads were then washed three times with Ni-binding buffer and 250 nM FHIP1B-HaloTag diluted in modified Ni-binding buffer (25 mM Tris–HCl, pH 7.5, 250 mM NaCl, 10 mM imidazole, 0.1% casein) was added to the beads. FHIP1B-HaloTag-conjugated bead complexes were gently shaken for 30 min at room temperature following three washes with 1 ml Ni-binding buffer. FHIP1B-HaloTag was eluted of the beads by applying 30 μl of elution buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 300 mM imidazole, 0.01% NP-40) and 10-min incubation at room temperature. The elution was analyzed via SDS–PAGE on a gel stained with SYPRO Red (Thermo Fisher Scientific).