Small ni nta agarose

A membrane protein detergent screen on AmtB-GFP was performed as previously described^{16 (link)} with minor modifications. Briefly, AmtB-GFP was extracted from purified membranes (described above) with 1-2% (w/v) of the detergent of interest in Buffer B and incubated for one to three hours at room temperature or overnight at 4 °C with gentle agitation. Insoluble material was pelleted by centrifugation at 20,000 g for 25 minutes at 4 °C. The clarified supernatant was loaded onto small Ni-NTA agarose (Qiagen) drip columns (Bio-spin Chromatography columns, Bio-Rad) and washed with several column volumes of Buffer C (200 mM sodium chloride, 20 mM imidazole, 5 mM BME, 50 mM Tris, pH 8.0 at room temperature) supplemented with two times the critical micelle concentration (CMC) of the detergent of interest. AmtB-GFP was eluted with two column volumes of Buffer D (100 mM sodium chloride, 250 mM imidazole, 5 mM BME, 2x CMC detergent of interest, and 50 mM Tris, pH 8.0 at room temperature). Eluted protein was concentrated using a 100 kDa Molecular Weight Cutoff (MWCO) concentrator and buffer exchanged into MS Buffer (2x CMC detergent of interest and 200 mM ammonium acetate, pH 8.0 with ammonium hydroxide) using a centrifugal buffer exchange device (Micro Bio-Spin 6, Bio-Rad).