Stronger binding to an IMAC column can be particularly useful when the recombinant his-tagged proteins are from a eukaryotic source. Most nonrecombinant proteins bind more weakly than a long string of histidines and so elute at a lower concentration of imidazole. Stronger binding can be an advantage when the crude protein mixture contains unwanted proteins that have a high affinity for the charged IMAC resin. As the number of histidine residues increases, the elution buffer requires a higher concentration of imidazole to remove bound proteins from the column. His-tag Purification Featuresįor his-tag purification, a longer his-tag or two hexahistidine tags provides a stronger affinity for the matrix. Uncharged kits give users the option of trying different metals to determine if one gives higher purity or yield for a particular his-tagged recombinant protein. Uncharged resins and kits that can be charged with Co 2+ or other divalent or trivalent metal ions are also available. Bio-Rad has his-tag resins and his-tag purification kits that are precharged with Ni 2+ for fast, easy his-tag protein purification. Using Co 2+ can give higher purity but with a lower yield. Ni 2+ is most commonly used for his-tag purification since it gives a high yield. His-tagged protein is then eluted with a higher concentration of imidazole. Typically, a low concentration of imidazole is added to both binding and wash buffers to interfere with the weak binding of other proteins and to elute any proteins that weakly bind. Imidazole competes with the his-tag for binding to the metal-charged resin and thus is used for elution of the protein from an IMAC column. The use of a his-tag and IMAC can often provide relatively pure recombinant protein directly from a crude lysate. Most other proteins in the lysate will not bind to the resin, or bind only weakly.

The his-tag has a high affinity for these metal ions and binds strongly to the IMAC column. The most common ion for his-tag purification of a recombinant protein is Ni 2+, though Co 2+, Cu 2+, and Zn 2+ are also used. In this technique, transition metal ions are immobilized on a resin matrix using a chelating agent such as iminodiacetic acid. His-tag purification uses the purification technique of immobilized metal affinity chromatography, or IMAC. Some recombinant proteins are engineered to have two hexahistidine tags.

There can be from four to ten residues in a string, although commonly there are six histidine residues - a hexahistidine tag. A his-tag, or polyhistidine tag, is a string of histidine residues at either the N or C terminus of a recombinant protein.

Expression media for mac