Pi mix hp

For the orthogonal purification of TAP complexes, all steps were carried out on ice and all buffers were adjusted to pH 7.4. The transfected and harvested cells of six 15 cm dishes were solubilized for 2 h in buffer 1 (20 mM HEPES/NaOH, 200 mM NaCl, 50 mM KCl and 15% (v/v) glycerol) supplemented with 10 mM imidazole, 1 × PI-mix HP (Serva), and 2% (w/v) glyco-diosgenin (GDN, Anatrace). The samples were centrifuged 30 min at 120,000 × g and 4 °C. The proteins were bound to 300 μl Ni²⁺-resin (Ni²⁺-Sepharose 6 Fast Flow, GE Healthcare) for 2 h. The beads were washed twice 15 min with buffer 1 supplemented with 10 mM imidazole and 0.05% GDN. To elute the proteins, the beads were incubated 30 min in buffer 1 supplemented with 200 mM imidazole and 0.05% GDN. The eluate was incubated for 3 h with 200 μl streptavidin resin (High Capacity Streptavidin Agarose Resin, Thermo Scientific). The beads were washed twice for 15 min with buffer 1 supplemented with 0.05% GDN. Subsequently, bound proteins were eluted for 45 min in buffer 1 supplemented with 2.5 mM biotin and 0.05% GDN. The eluate was frozen in liquid nitrogen and stored at −80 °C.

For the orthogonal purification of coreTAP complexes, all steps were carried out on ice, and all buffers were adjusted to pH 7.4. The transiently transfected and harvested HEK293-F cells were solubilized for 1.5 h in buffer 1 (20 mM HEPES/NaOH, 200 mM NaCl, 50 mM KCl, and 15% (v/v) glycerol) supplemented with 10 mM imidazole, 1× PI-mix HP (Serva), and 2% (w/v) glyco-diosgenin (GDN, Anatrace). The samples were centrifuged for 30 min at 120,000 × g and 4 °C. The proteins were bound to 200 μl Ni-NTA Sepharose 6 Fast Flow (GE Healthcare) for 2 h. The beads were washed twice for 15 min with buffer 1 supplemented with 10 mM imidazole and 0.05% GDN. To elute the proteins, the beads were incubated for 30 min in buffer 1 supplemented with 200 mM imidazole and 0.05% GDN. The eluate was incubated 3 h with 200 μl high-capacity streptavidin agarose resin (Thermo Fisher Scientific). The beads were washed twice for 15 min in buffer 1 supplemented with 0.05% GDN. Subsequently, bound proteins were eluted for 45 min in buffer 1 supplemented with 2.5 mM biotin and 0.05% GDN. The eluate was frozen in liquid nitrogen and stored at −80 °C.