Ni-NTA Superflow Columns enable up to 24 large-scale 6xHis-tagged protein preparations to be performed in parallel on the BioRobot® 3000 workstation. The pre-packed columns are pre-charged with nickel and can also be used for manual, gravity-flow purification. Using cleared lysates as starting material, up to 15 mg protein per column can be purified under native or denaturing conditions. For protein structure analyses by NMR or X-ray diffraction, a single Ni-NTA Superflow Column can provide enough highly pure protein for a comprehensive functional and structural analysis.