Non-biotinylated proteins in the supernatants were removed and concentrated by spin filtration using columns with 30 kDa molecular weight cutoff (Millipore).
Purification was performed by chromatography on S-Sepharose Fast Flow (Pharmacia, Uppsala, Sweden), or Mono S 5/5 column (Pharmacia), followed by filtration using Centriplus 100 (Amicon, Beverly, MA).
Labeled antibodies were purified from free dye by gel filtration using a column provided in the kit.
After the lipid solution was extruded, free calcein was removed by gel filtration using a column (1 cm ID, 15 cm in length) packed with Sephadex G-200 (superfine) resin (GE Healthcare).
Next, ion exchange chromatography using HiTrap SP HP column (GE Healthcare, Germany) connected to AEKTA purifier (GE Healthcare) was applied followed by gel filtration using S75 16/60 column (GE Healthcare) as the final step.
Also, unless otherwise noted, the protein was further purified by ion-exchange chromatography, followed by gel filtration using a Superdex200 column (GE Healthcare), equilibrated with 50 mM HEPES, pH 7.5, 200 mM KCl, 1 mM DTT, and 1 mM TCEP.
KIF5B-Atto550 was removed from free dye by gel filtration using a NAP5 column.
The VSG was further purified by gel filtration using Sephacryl S200 column (4 × 90 cm) equilibrated with 0.1 M NH4HCO3.
The first three eluted fractions of 1 ml known to have the highest PmHS2 concentrations were pooled and then desalted by gel filtration using a PD10 column with Sephadex G-25 Medium resin (GEA Healthcare) with 50 mM Tris pH 7.2 as eluent.
Excessive NEM was removed by gel filtration using PD-10 columns.
