Ni-NTA Superflow 96 BioRobot Kit
For automated, medium-scale purification of 6xHis-tagged proteins
- Fully automated purification of up to 10 mg protein per well
- High-purity protein free from cross-contamination
- Purification under native or denaturing conditions
The Ni-NTA Superflow 96 BioRobot Kit provides automated purification of up to 600 µg recombinant protein from 96 samples in parallel (standard protocol, 5 ml culture volume per sample). Cleared bacterial cell lysates flow onto a filter plate preloaded with Ni-NTA Superflow by the BioRobot workstation. This unique 96-well metal-chelate affinity-chromatography module strongly and selectively binds His-tagged proteins. Ready-to-run protocols are provided for purification under native or denaturing conditions.
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Ni-NTA Superflow 96 BioRobot Kit (4)
For 4 x 96 6xHis-tagged protein preps: 4 QIAfilter 96 Plates, 4 TurboFilter 96 Plates, 1 x 100 ml Ni-NTA Superflow
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969261
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The Ni-NTA Superflow 96 BioRobot Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Protein purification with the Ni-NTA protein purification system.|Ni-NTA Superflow 96 BioRobot Kit.|Automated expression clone screening.|Milligram amounts of pure His-tagged proteins per well.|
||Medium-scale purification for expression-clone screening and protein characterization. A mixture of vector constructs for the expression of proteins of 12, 15, and 28 kDa, and a 48/50 kDa heterodimer was transformed into E. coli, plated on selective medium, and 96 colonies were picked at random for inoculating 5 ml cultures. Expression of 6xHis-tagged proteins was induced with IPTG overnight. Cells were pelleted and lysed and 6xHis-tagged proteins purified. 5 µl of each elution fraction was loaded for SDSPAGE analysis; proteins were visualized by Coomassie staining.
|Vector constructs for the expression of His-tagged proteins were transformed into E. coli, plated on selective medium, and colonies were picked for inoculating 25 ml cultures. Expression of 6xHis-tagged proteins was induced with IPTG for 2–4 hours. Cells were pelleted in 24-well blocks and processed on the BioRobot 3000 using 200 µl Ni-NTA Superflow resin per well. 5 µl (0.9%) of the first elution fraction was loaded for SDS-PAGE and proteins were visualized by Coomassie staining. G: Green Fluorescent Protein (29 kDa); T: T7 RNA Polymerase (100 kDa); S: E. coli GroES (12 kDa). Some endogenous GroEL is copurified; C: E. coli chloramphenicol acetyltransferase (28 kDa); L: E. coli GroEL (60 kDa); α: human tumor necrosis factor α(18 kDa); E: E. coli GroES purified as a complex with co-overexpressed nontagged GroEL (12 and 60 kDa); 10: Saccharomyces cerevisiae Cpn-10 (10 kDa). M: markers.|
Performance
In response to customers’ needs for larger amounts of protein from automated procedures, QIAGEN has adapted the Ni-NTA Superflow 96 BioRobot protocol to enable processing of larger culture volumes and purification of milligram amounts of His-tagged protein per well. Increasing the amount of Ni-NTA Superflow resin used in the procedure to 200 µl per well and an optimized lysis buffer formulation enables up to 25 ml (purification under native conditions) or 15 ml (purification under denaturing conditions) cultures to be processed (high-yield protocols). Cell cultures are transferred to 24-well blocks for processing. The corresponding increase in biomass can deliver in some cases up to 10 mg of pure His-tagged protein per well (see table and figure Milligram amounts of His-tagged proteins per well) allowing multiple assays to be carried out on the same batch of protein and reducing the total number of protein preps required, see also figure Automated expression clone screening. The single-step purification provides highly pure proteins over a wide range of yields, and purification of large protein complexes is possible. | Green fluorescent protein | 4000 | 6.0 | | T7 RNA polymerase | 1000 | 1.4 | | GroES | 300 | 0.4 | | Chloramphenicol acetyltransferase | 2400 | 4.4 | | GroEL | 740 | 1.0 | | Tumor necrosis factor α | 1600 | 2.5 | | GroES/GroEL | 1200 | 1.5 | | Cpn-10 | 170 | 0.3 |
Principle
The QIAexpress Ni-NTA Protein Purification System, including the Ni-NTA Superflow 96 BioRobot Kit, is based on the remarkable selectivity of patented Ni-NTA (nickel-nitrilotriacetic acid) resin for proteins containing an affinity tag of six or more histidine residues — the His tag. This technology allows one-step purification of almost any His-tagged protein from any expression system under native or denaturing conditions. NTA, which has four chelation sites for nickel ions, binds nickel more tightly than metal-chelating purification systems that only have three sites available for interaction with metal ions. The extra chelation site prevents nickel-ion leaching and results in a greater binding capacity and protein preparations with higher purity than those obtained using other metal-chelating purification systems. The QIAexpress system can be used to purify His-tagged proteins from any expression system including baculovirus, mammalian cells, yeast, and bacteria. (See figure Protein purification with the Ni-NTA protein purification system).
Procedure
The purification of His-tagged proteins consists of 4 stages: cell lysis, binding, washing, and elution anfd is the basis of the Ni-NTA Superflow BioRobot Kit (see figure Ni-NTA Superflow 96 BioRobot Kit). Purification of recombinant proteins using the QIA express system does not depend on the 3-dimensional structure of the protein or 6xHis tag. This allows one-step protein purification under either native or denaturing conditions, from dilute solutions and crude lysates. Strong denaturants and detergents can be used for efficient solubilization and purification of receptors, membrane proteins, and proteins that form inclusion bodies. Reagents that allow efficient removal of nonspecifically binding contaminants can be included in wash buffers (see table). Purified proteins are eluted under mild conditions by adding 100–250 mM imidazole as competitor or by a reduction in pH. The Ni-NTA Superflow 96 BioRobot Kit is for use with BioRobot 3000, 8000, or 9600 workstations. .
| 6 M Gu·HCl | 2% Triton X-100 | 20 mM β-ME | 50% glycerol | 4 M MgCl2 | Up to 30% ethanol | | 8 M urea | 2% Tween 20 | 10 mM DTT | 20% ethanol | 5 mM CaCl2 | or 100 mM NaOH | | | 1% CHAPS | 20 mM TCEP | 20 mM imidazole | 2 M NaCl | |
Applications
The QIAexpress Ni-NTA Protein Purification System provides reliable, one-step purification of proteins suitable for many application, including: - Structural and functional investigations
- Crystallization for determination of three-dimensional structure
- Assays involving protein–protein and protein–DNA interactions
- Immunization to produce antibodies
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The two protocols given below are for the use of the Ni-NTA Superflow 96 BioRobot® Kit in manual procedures. The kit has been specially designed and optimized for automated 6xHis-tagged protein purification on QIAGEN® BioRobot Systems. For more details of the advantages of BioRobot Systems see the Ni-NTA Superflow 96 BioRobot Kit Handbook supplied with the kit or contact one of the QIAGEN Technical Service Departments or local distributors listed on the last page of the handbook.
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The following protocols have been designed for the use of Ni-NTA Superflow Columns on the QIAvac 6S vacuum manifold or in gravity-flow applications on the QIArack. Up to 15 mg 6xHistagged protein can be purified per column from cleared lysate derived from up to 1 liter of (E. coli) bacterial culture.
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