Histrap hp ni nta

E. coli (JE28) cells⁵⁰ were grown in M9 media containing ¹⁵N NH₄Cl (1 g/l) and 30 µg/ml kanamycin at 37 °C. At an OD_600nm of around 0.8, the cells were cooled on ice for 15 min and harvested by centrifugation (5000 × g, 30 min, 4 °C). Cells were lysed and purified by affinity chromatography as described before⁵⁰ . Briefly, the cells were harvested by centrifugation (5000 × g, 30 min, 4 °C) and were disrupted using a M-100P microfluidizer (1000 bar, Microfluidics, Westwood, USA) in ribosome buffer (20 mM Tris-HCl pH 7.6, 10 mM MgCl₂, 150 mM KCl, 30 mM NH₄Cl). The lysate was centrifuged twice (38,400 × g, 4 °C, 30 min) and further purified using nickel-affinity chromatography (HisTrap HP, Ni-NTA) (GE Healthcare, Chicago, USA) equilibrated with ribosome buffer. The ribosomes were eluted in a linear gradient to 150 mM imidazole. Ribosome-containg fractions were pooled and loaded on a 10–40% (w/v) sucrose gradient. The buffer was exchanged to JE28 buffer (20 mM Tris-HCl, 10 mM MgCl₂, 150 mM KCl, 30 mM NH₄Cl, pH 7.6). The final sample was concentrated to 11 µM for the DNP-enhanced solid-state NMR experiments. ¹⁵N-labeled ribosomes were used to optimize the glycerol cushions required for the solid-state NMR experiments.