S200 16 60 column

Sequences coding for H6-BubR1, H6-Bub1, and untagged Bub3 constructs were sub-cloned into pFLMultiBac vectors and baculoviruses were generated. Baculovirus expressing Bub3-TRX was generated by the Dortmund Protein Facility (DPF) using the pOPIN vector system {Berrow:2007cy}. Bub1/Bub3 and BubR1/Bub3 complexes were generated by co-infection and co-expression at 27°C for 72 hr. Insect cells were harvested by centrifugation at 1500 rpm for 30 min in a Sorvall RC 3BP+ (Thermo Scientific) centrifuge with Rotor H6000A, the pellets were frozen in liquid nitrogen and stored at −80°C. 1 g of cell pellet was re-suspended in 10 ml Lysis buffer (50 mM HEPES-KOH pH 7.5, 150 mM KCl, 15 mM imidazole, 2 mM DTE, 0.05% Tween20, PMSF, protease inhibitors [Serva]). Cells were lysed by sonication and centrifuged at 100000×g for 1 hr at 4°C. The supernatant was filtered through Nalgene bottle-top filter. The complexes were isolated from the cleared lysate on a 5-ml HisTrap (GE Healthcare) column. Peak fractions were pooled concentrated using Amicon concentrators and further purified in GF buffer (50 mM Hepes-KOH pH 7.5, 150 mM KCl, 2 mM DTE, 0.05% Tween20) by size exclusion chromatography using S200 16/60 column (GE Healthcare). Peak fractions were pooled, concentrated to typically 3 to 5 mg/ml, and frozen in liquid nitrogen.