The indicated proteins were purified in duplicate under native conditions using Ni-NTA Spin Columns from cleared E. coli cell lysates derived from 5 ml LB cultures either manually or in an automated procedure on the QIAcube. CAT: chloramphenicol acetyl transferase; GFP: Green fluorescent protein; HIV-RT: Human immunodeficiency virus reverse transcriptase; IL-1b: Interleukin-1 beta. M: markers; C: cleared lysate (2 μl loaded per lane); E: elution fraction (3 μl loaded per lane).
The 6xHis-tagged mouse DHFR was expressed at the indicated levels in E. coli, purified from 3 ml cultures using the Ni-NTA Spin Kit under denaturing conditions, and eluted in buffer at pH 5.9. Fractions were visualized by Coomassie staining after SDS-PAGE. 5 µl (of each 200 µl eluate) was loaded. 1: cell lysate; 2: flow-through 3: first eluate; 4: second eluate.