a HISTrap HP column (GE Healthcare) equilibrated in wash buffer (50 mM HEPES, pH 7.0; 150 mM KCl; 0.4% DDM; 20 mM imidazole) connected to an ÄKTA pure (GE Healthcare) chromatography system and eluted in buffer containing 300 mM imidazole. The HISTrap column eluate was pooled, exchanged into imidazole-free buffer and digested overnight at 4 °C with HRV 3 C protease, purified as described71 (a generous gift of Daniel Minor, UCSF, San Francisco, CA). Digested protein was concentrated and run on a Superdex-200 gel filtration column in 20 mM TRIS-HCl pH 7.5, 150 mM KCl, 0.1% (w/v) DDM (anagrade), 5 mM DTT, and 1 mM EDTA. Fractions eluting at a volume consistent with the GIRK channel tetramer were pooled, concentrated and examined by SDS-PAGE and Coomassie blue staining. We also analyzed the purified protein using tandem mass spectrometry and detected significant peptides for only GIRK2 and the mass spectrometry experimental artifacts trypsin and keratin. No other proteins were evident.
