Ni nta histrap ff column

Total extract of CHO cells expressing the β₁ 6His construct were immobilized with nickel-nitrilotriacetic acid beads (Ni⁺-NTA, His Trap FF column; GE Healthcare, Chicago, IL, USA) equilibrated with 10 mL of RIPA buffer containing protease inhibitors. Total protein extracts (500 µg) were loaded into the resin, and allowed to interact for 3 h at 4 °C with gentle shaking. Then the unbound protein was washed as indicated by the manufacturer, and the total extract of CHO β₁ YFP cells incubated in the presence or absence of ouabain (50 nM and 100 µM) were loaded as a prey. Samples were incubated overnight at 4 °C, and washed with PBS supplemented with 10 and 20 mM imidazole. Interacting proteins were eluted in PBS containing 500 mM imidazole and were analyzed by Western blot.