The purification of 6xHis-tagged proteins consists of 4 steps: cell lysis, binding, washing, and elution (see " Protein purification with the Ni-NTA protein purification system
"). 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 "Reagents compatible with the 6xHis/Ni-NTA interaction
"). Purified proteins are eluted under mild conditions by adding 100–250 mM imidazole as competitor or by a reduction in pH.
| 6 M Gu·HCl || 2% Triton X-100 || 20 mM β-ME || 50% glycerol ||4 M MgCl2 ||Up to 30% ethanol or 100 mM NaOH |
| 8 M Urea || 2% Tween 20 || 10 mM DTT || 20% ethanol ||5 mM CaCl2 || |
| || 1% CHAPS || || 20 mM imidazole ||2 M NaCl || |