Cnbr activated sepharose resin

To purify the flWT-Kv3.1a tetramer, cells were resuspended in buffer A (Tris 20 mM pH 7.5, KCl 200 mM, MgCl2 1 mM, Lauryl Maltose Neopentyl Glycol 1%, cholesteryl hemisuccinate 0.1%, Protease inhibitor cocktail EDTA free from Roche according to manufacturer recommendations, and DNASE from Roche at 0.01 mg/ml). Cells were homogenized and the flWT-Kv3.1a tetramer was extracted by gentle stirring for 2 hours at 4°C. The nonsoluble fraction was removed by ultracentrifugation at 42,000 rpm at 4°C for 1 hour using Ti45 Beckman rotor. The soluble fraction was mixed with noncommercial Pro-GFP resin (anti-GFP binder coupled to CNBr Activated Sepharose resin from GE-healthcare) pre-equilibrated with buffer B (Tris 20 mM pH 7.5, KCl 200 mM, Lauryl Maltose Neopentyl Glycol 0.01%, and DTT 1 mM). After incubation for 2 hours at 4°C, the resin was washed extensively with buffer B and the flWT-Kv3.1a tetramer was eluted by addition of 100 µl of HRV 3C Protease at 1 U/µl concentration from Takara Bio. Elution was performed overnight at 4°C. Eluted protein was concentrated on vivaspin 100 kDa cutoff Vivaspin Turbo 15 concentrator and injected on a 10/30 Superose 6 size exclusion chromatography column pre-equilibrated with buffer B. Fractions, which correspond to the tetramer were pooled and concentrated to reach 1 mg/ml concentration.

For Fig. 4b, we conducted a pull-down assay using immobilized lamin 300 A146C mutant protein. The lamin 300 A146C mutant protein was coupled to CNBr-activated Sepharose resin (GE Healthcare) and/or blocked with excessive 0.1 M Tris-HCl (pH 8.0) buffer containing 150 mM NaCl. Four purified lamin fragments (residues 1–125, 175–300, 250–400, and 406–567; 66 μM) were incubated with the lamin-coupled resin or Tris-blocked resin (for control), which were pre-equilibrated with a 20 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl. After washing with the buffer, the resin was analysed using SDS-PAGE.
For Fig. 4c, His-tagged lamin proteins were immobilized on the Ni-NTA resin as bait. BSA or His-tag cleaved lamin proteins as a prey were incubated on the His-lamin immobilized resin pre-equilibrated in a 20 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl (or 50 mM NaCl) at room temperature for 30 min. After washing with the buffer supplemented with 20 mM imidazole, the resin was analysed using SDS-PAGE.