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 QIAexpress
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 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 || |