Histrap hp affinity chromatography

pET28a-His₆-Get4 and pETcoco2-Get5 were transformed into E. coli BL21(DE3) and expression was induced at 25 °C with 2 mM isopropyl-β-d-1-thiogalactopyranoside. The His₆Get4/Get5 complex was then purified using Ni-NTA super flow beads according to the manufacturer’s manual under native conditions (QIAGEN) (Supplementary Fig. 4i). Purified His₆Get4/Get5 was transferred to Hepes buffer (20 mM Hepes/KOH pH 7.4, 2 mM Mg(OAc)₂, 120 mM KOAc, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF)) using PD-10 desalting columns (GE Healthcare) and was then employed in the experiments shown in Figs. 4 and 5. Get4/His₆Get5 was expressed in E. coli BL21(DE3) from plasmid pETDuet-His₆Get5-Get4. Purification of Get4/His₆Get5 of was performed using HisTrap HP affinity chromatography (GE Healthcare) followed by size-exclusion chromatography (Superdex 200 10/300 GE Healthcare). Purified Get4/His₆Get5 was transferred to 50 mM Tris/HCl pH 7.5, 70 mM NH₄Cl, 30 mM KCl, 7 mM MgCl₂ buffer using PD-10 desalting columns (GE Healthcare) prior to fluorescent labeling for anisotropy and FCS measurements shown in Fig. 1. Purified proteins were stored at −80 °C.