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Ni-NTA Superflow Columns - automated large-scale purification of 6xHis-tagged ,,, proteins

TA prevents nickel-ion leaching, increasing protein binding capacity and minimizing nonspecific binding.

Automated procedure for large-scale purification

Ready-to-run protocols for automated purification under native or denaturing conditions have been developed. Columns, prepacked with Ni-NTA Superflow (1.5 ml bed volume), are placed on the vacuum manifold of the BioRobot 3000 workstation. Up to 12 columns can be processed per manifold. Cells from up to 1 liter of bacterial culture are lysed using a buffer containing nucleases, and cleared lysates are prepared by centrifugation. Cleared lysates are transferred to 14 ml tubes and placed with the relevant number of Ni-NTA Superflow Columns on the BioRobot worktable for processing. The automated procedure then begins. After column equilibration, the BioRobot 3000 workstation pipets cleared lysates onto
Ni-NTA Superflow Columns. 6xHis-tagged proteins bind strongly and selectively to Ni-NTA while contaminants are removed in the flow-through fractions. After 2 wash steps, highly pure 6xHis-tagged proteins are eluted into 24-well blocks using 3 ml of elution buffer. Lysates, wash fractions, and pure proteins are drawn through Ni-NTA Superflow Columns by applying a vacuum (see flowchart).

High yields and high reproducibility

The Ni-NTA Superflow Column procedure was developed to address customers needs for large amounts of highly pure protein. Typically, 510 mg of a protein is needed to determine its structure by NMR or X-ray crys
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