Purification and Labeling of Tubulin and CENP-F Fragments

Reagents were purchased from Sigma-­Aldrich unless stated otherwise. Tubulin and CENP-F fragments were purified as described (Volkov et al., 2015 (link)). In brief, tubulin was purified from cow brains by cycles of polymerization in 0.33 M 1,4-piperazinediethanesulfonic acid (PIPES) and depolymerization in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) and 1 mM CaCl₂ as described (Castoldi and Popov, 2003 (link)). Cycled tubulin was then polymerized and additionally labeled with succinimidyl esters of 5-carboxytetramethylrhodamine (Thermo­Fisher), HiLyte-647 (Anaspec), or DIG (ThermoFisher) as described (Hyman et al., 1991 (link)). CENP-F fragments were expressed in Rosetta cells under induction by 100 mM isopropyl-β-d-thiogalactoside for 2 h at 37°C. Cells were lysed with ultrasound or B-PER reagent (2592C and 2873C; ThermoFisher), and CENP-F fragments were purified on Ni–nitriloacetic acid (NTA)–agarose beads (Qiagen), desalted, and then applied on a prepacked HiTrap column (GE Healthcare). Protein was eluted from HiTrap columns with a linear gradient from 0.1 to 1.0 M NaCl in sodium phosphate buffer at pH 7.0 (or 7.2 for sfGFP-2892C and sfGFP-2927C). sfGFP-3003C and sfGFP-2892CΔ111 were purified using single-step Ni-NTA purification. Peak fractions were determined by SDS–PAGE, aliquoted in 30% glycerol, snap-frozen in liquid nitrogen, and stored at −80°C.