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

Ni-NTA Superflow Columns enable up to 24 automated large-scale 6xHis-tagged protein preparations to be performed in parallel on the BioRobot 3000 workstation. Using cleared lysates from bacterial cultures as starting material, up to 15 mg protein per column can be purified under native or denaturing
conditions using ready-to-run protocols. The increase in yield means that more experiments can be performed using a single batch of protein, with enough protein for a structural determination being obtained from a single column.

Ni-NTA Superflow Columns offer:
  • High yields - up to 15 mg highly pure 6xHis-tagged protein per column
  • Automated purification - multiparallel processing of up to 24 samples on the BioRobot 3000 workstation
  • High purity - cross-contaminationCfree processing with high column-to-column reproducibility
  • Convenience - ready-to-run protocols for purification under native or denaturing conditions
Purification using proven 6xHis-tagNi-NTA technology Ni-NTA Superflow Columns
Automated Procedure
6xHis-tagged proteins are efficiently purified on Ni-NTA Superflow Columns using the highly selective interaction between an affinity tag of 6 consecutive histidine residues (the 6xHis tag) and nickel-nitrilotriacetic acid (Ni-NTA). The four chelating sites of NTA bind nickel ions more tightly than alternative metalchelation purification systems that use ligands with only 3 chelating sites. The extra chelating site of N 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 tallography. Currently, methods like FPLC are used for the purification of such amounts. The Ni-NTA Superflow Column procedure enables automated purification of up to 24 samples each containing up to 15 mg 6xHis-tagged protein (Table 1). Figures 1 and 2 show the high reproducibility and absence of cross-contamination in the automated Ni-NTA Superflow Column purification procedure. Ni-NTA Superflow Columns can also be used for manual gravity-flow purification of 6xHistagged proteins.

Cross-ContaminationFree Processing Highly Reproducible Yields Figure 1 SDS-PAGE analysis of twelve 6xHis-tagged protein preparations performed in parallel using Ni-NTA Superflow Columns. For E. coli culture volumes and yields see Table 1. CAT: 6xHis-tagged chloramphenicol acetyl-transferase; TNF- alpha: 6xHis-tagged tumor necrosis factor- alpha; 46: 6xHis-tagged 46 kDa protein; 38: 6xHistagged 38 kDa protein; GFP: 6xHis-tagged green fluorescent protein; 42: 6xHis-tagged 42 kDa protein. Aliquots (5 l) of the final eluates (3 ml) were loaded onto the gel. Proteins were visualized by Coomassie
staining. M: markers. Figure 2 SDS-PAGE analysis of twelve 6xHistagged green fluorescent protein preparations. Cleared lysates from 100 ml E. coli cultures were processed in parallel using Ni-NTA Superflow Columns. Aliquots (5 l) of the final eluates
(3 ml) were loaded onto the gel. Proteins were visualized by Coomassie staining. M: markers.

Table 1. Protein yields from preparati ons performed using Ni-NTA Superflow Columns
Scalable automated protein purification

QIAGEN offers automated solutions for each stage of proteomics, functional genomics, and protein structure determination projects. Initial expression-clone screening can be performed using fast, fully automated Ni-NTA Magnetic Agarose Bead protocols. The Ni-NTA Superflow 96 BioRobot Kit enables
high-throughput purification of up to 2 mg 6xHis-tagged protein per well, sufficient for initial functional studies. 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.



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