Description
The Ni-NTA Superflow 96 BioRobot Kit provides automated purification of up to 300 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.
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 up to 2 mg 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 an increased yield 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. Yields of His-tagged proteins using the adapted Ni-NTA Superflow 96 BioRobot protocol His-tagged protein Total yield per well (µg)* Protein concentration (mg/ml)† 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 * Yield obtained in two 550 µl elution fractions (average of 6 independent purifications). 80% of the protein elutes in the first 550 µl. † Protein concentration in the first 550 µl elution fraction.
The QIA express 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 QIA express 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).
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. Reagents Compatible with the His/Ni-NTA Interaction Denaturants Detergents Reducing agents Others Salts For long-term storage 6 M Gu·HCl 2% Triton X-100 20 mM β-ME 50% glycerol 4 M MgCl 2 Up to 30% ethanol 8 M urea 2% Tween 20 10 mM DTT 20% ethanol 5 mM CaCl 2 or 100 mM NaOH 1% CHAPS 20 mM TCEP 20 mM imidazole 2 M NaCl
