Protein purification in LMNG/CHS

The isolated membranes were thawed on ice and solubilized by the addition of a mixture of detergents (lauryl maltose neopentyl glycol (LMNG) with cholesteryl hemisuccinate (CHS) in a 5 to 1 ratio) to a final concentration of 1% (w/v), and gently agitated for 2 hours at 4°C. The solution was cleared by ultracentrifugation (40 min at 1700 xg). Pre-equilibrated Ni²⁺-NTA resin (QIAGEN) was added to the sample, together with 10 mM imidazole and the mixture allowed to gently rotate overnight at 4°C. After transferring the mixture to a column, the resin was washed in two steps: 1) 5 column volumes of buffer containing 50 mM imidazole; 2) 5 column volumes of buffer containing 75 mM imidazole. The proteins were eluted with 10 column volumes of the following buffer: 200 mM NaCl, 20 mM HEPES, pH 7.5, 300 mM imidazole. The eluted protein was loaded on a Superose 6 increase 10/300 GL SEC column (GE Healthcare Life Sciences) pre-equilibrated with buffer containing 200 mM NaCl, 20 mM HEPES pH 7.0 and detergent (LMNG-CHS) at the concentration of 0.002% (w/v). For the protein sample used to solve the structure of HCN4 bound to cAMP, the ligand (Sigma-Aldrich) was kept at a concentration of 0.2 mM in all steps of membrane isolation and protein purification procedure described above. The decahistidine-eGFP tag at the N terminus of HCN4 protein was not removed. Final yield of purified protein was about 1mg per 1 l of cells.