Description

FastSep high performance resins and chromatography cartridges

- Numerous ligand-metal ion combinations 
- High affinity & high capacity 
- Minimal non-specific binding & excellent purity 
- Long spacer arms & minimal steric hindrance 
- Low metal ion leaching & excellent stability 
- Maintain high performance after multiple repeated use 

Application

- For His-tagged protein purification 
- Simply replace your existing Ni-NTA resin or cartridge, no optimization or protocol changes necessary 
- Purification under native and denaturing conditions 
- Suitable for small proteins, large protein complexes, and proteins with low expression rates 

Purification of His-tagged Recombinant Proteins

A his-tag, or polyhistidine tag, is a string of histidine residues at either the N or C terminus of a recombinant protein. Such a string can have 4 to 10 residues, with 6 being the most common a hexahistidine tag. Recombinant proteins can have more than one hexahistidine tag. 

In IMAC, metal ions are immobilized on a resin matrix using a chelating agent such as NTA or IDA. Ni2+, Co2+, Cu2+, and Zn2+ are the most commonly used ions for his-tag purification of a recombinant protein. The his-tag has a high affinity for these metal ions and binds strongly to IMAC column, while most other proteins in the lysate will not bind to resin, or bind only weakly. The use of a his-tag and IMAC can therefore provide relatively pure recombinant protein directly from a crude lysate. 

How to choose different ligands and metal ions in IMAC?

NTA and IDA are the two most widely used ligands in IMAC. NTA is a tetravalent ligand with coordination number 4 and therefore stronger coordination of metal ions, while IDA is a trivalent ligand with coordination number 3. Generally, NTA gives higher binding specificity and lower metal ion leaching, while IDA shows more non-specific binding and increased metal ion leaching. IDA can have higher metal ion loading density and usually requires much lower imidazole concentration in eluent. 

For metal ions going from Co2+, Zn2+, Ni2+, to Cu2+, generally binding affinity increases while binding specificity decreases. Cu2+ has the highest affinity giving the highest protein recovery, but low specificity. Co2+ has the lowest affinity but highest specificity, reducing the amount of impurity proteins in eluate. Ni2+ is often most widely used because it provides a good balance between affinity and specificity. 

As many factors can affect the choice of optimal ligand-metal ion combination, the best approach is to try different combinations and let your proteins decide. FastSep(TM) high performance resins and cartridges offer numerous ligand-metal ion combinations to meet various purification needs. 
FastSep(TM) high performance resins and chromatography cartridges are based on highly cross-linked agarose 6% beads which have structural characteristics that result in superb physical strength, excellent flow properties, low backpressure, and an open pore structure. FastSep(TM) high performance resins and chromatography cartridges allow operation at very high flow rates, resulting in increased throughput and rapid column cleaning between chromatography runs. 

Specifications