Ds 11 microvolume spectrophotometer

The wild type MgtE was expressed in 1 L cultures and induced at 37 °C, 30 °C and 20 °C, respectively. The harvested cells were disrupted and the membrane fraction was solubilized using different detergents as described above. The supernatant containing the detergent-solubilized membrane was incubated with Ni²⁺ resin for 15 min. followed by passing it through a column. The resin was washed with 20 mM HEPES, 150 mM NaCl, 1 mM DDM, 50 mM imidazole, pH 7.0 buffer and MgtE was eluted with buffer containing 300 mM imidazole and 1 mM DDM. The eluate was then concentrated in Amicon Ultra 30K filter (Merck Millipore). To analyze whether the channel is properly folded, the purified protein was applied onto a Superdex 75 10/300 column (GE Healthcare) size-exclusion column equilibrated with 20 mM HEPES, 150 mM NaCl, 1 mM DDM, pH 7.0 buffer. The main peak of the gel filtration profiles was collected to measure the concentration of the purified MgtE (normalized to per litre of culture) using the molecular weight (~50 KDa) and molar extinction coefficient (53860 M^-1cm^-1) of wild-type MgtE in a DS-11+ microvolume spectrophotometer (DeNovix).