Fang-Fang Wu, Xuemei He, and David R Nau, Bio-Rad Laboratories, Inc., Hercules, CA 94547 USA
Immobilized metal affinity chromatography (IMAC) is a powerful technique that can be used for the efficient purification of recombinant histidine-tagged (His-tagged) proteins from a variety of expression systems.
The synthesis of IMAC resins begins with the derivatization of an appropriate wide-pore base resin with metal chelating groups, such as iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA). Specific transition metal ions usually Cu2+, Ni2+, Co2+, Ca2+, or Zn2+ are then immobilized to form a charged support. IMAC resins exhibit high affinity and metal-dependent selectivity for His-tagged proteins, as well as naturally occurring proteins that are rich in histidine, cysteine, aspartic acid, or glutamic acid residues.
To obtain highly purified proteins from an IMAC system, a series of method development experiments is typically required. These experiments involve optimization of the mobile phase pH, buffer system, flow rate, concentration of the competitive eluting agents, and a number of other potential parameters that may also be examined, depending on purity and recovery requirements. Usually, these would require a series of tedious and repetitive operations by the researcher, including the loading and recharging of the column with fresh metal ions after every run in order to ensure reproducible results.
Bio-Rad has developed an extremely rapid and efficient
method using the BioLogic DuoFlow high-resolution
chromatography system to purify a 75 kD His-tagged protein