Suitable for gravity flow and batch purification
His-tagged proteins are efficiently purified by a one-step procedure from crude lysates both under denaturing and non-denaturing conditions.
Product characteristics | |
|---|---|
| Matrix | 6 % cross-linked agarose |
| Bead size | 50-150 µm |
| Linker | Iminodiacetic acid (IDA) |
| Metal loading capacity | Low density: 5-20 µmol Me2+/ ml resin High density: 20-40 µmol Me2+/ ml resin |
| Linear flow rate | 26 cm/h |
| Maximum pressure | 0.18 bar (2.6 psi) |
| pH stability | 2-12 |
| Chemical stability | Stable to all solutions commonly used in gel filtration including 8 M urea and 6 M guanidine hydrochloride |
Based on the HSAP concept, IDA-immobilized Cu2+, Ni2+, Zn2+ and Co2+ ions exhibit different affinities & specificities towards histidines [2,3]. Copper (Cu2+): Shows the lowest specificity resulting in high target recoveris. Unspecific protein binding is minimized by low density metal loading. Cobalt (Co2+): Shows the highest binding specificity resulting in reduced unspecific protein binding. Target loss is minimized by high density metal loading. Nickel (Ni2+) and Zinc (Zn2+): Show intermediate selectivity. While using Ni2+ ions is the standard method, IDA-immobilized Zn2+ may prove superior to either immobilized Cu2+ and Ni2+ ions, as a result of its relatively low binding affinity for E. coli host cell proteins[4]. Low density metal loading enhances the qualitative purification of recombinant protein but with lower target recoveries. High density metal loading results in greater purification of recombinant proteins. However, unwanted proteins within the sample will also be bound.
[1] Porath et al. (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258:598.
[2] Gaberc-Porekar et al. (2001) Perspectives of immobilized-metal affinity chromatography. J. Biochem. Biophys. Methods 49:335.
[3] Ueda et al. (2003) Current and prospective applications of metal ion–protein binding. Journal of Chromatography 988:1.
[4] Richard et al. (2000) Design of Affinity Tags for One-Step Protein Purification from Immobilized Zinc Columns. Biotechnol. Prog. 16:86.
