Your histidine-tagged (his-tagged) purification and analysis workflow includes sample preparation, filtration, purification, purity check and Western blotting for protein identification and/or quantitation. Each of the steps and products selected will influence the results in terms of recovery, purity and analytical quality but will also open opportunities to save time and money.
Use of IMAC for his-tagged protein purification - What is IMAC?
IMAC is based on the interaction of proteins with certain amino acid residues on their surface and divalent metal ions (e.g., Ni2+, Cu2+, Zn2+, Co2+) immobilized via a chelating ligand. The interaction is primarily between histidine and metal ions, but also, for example, tryptophan and cysteine. His- tagged proteins have extra high affinity in IMAC because of the multiple (6 to 10) histidine residues. These proteins are usually the strongest binder among all the proteins in a crude sample extract (e.g., a bacterial lysate), while other cellular proteins will not bind or will bind weakly.
What do his-tagged protein purification schemes look like?
Below you will find some recommendations for purification of his-tagged proteins that can be considered when planning your experiment. The choice of techniques and combination will depend on needed purity and yield for your protein of interest.
Please carefully define your objectives and consider that in general every added purification step (except for buffer exchange) will increase purity but decrease total protein recovery and yield. In the picture below (Fig. 2), we describe typical proven techniques combination for the purification of his-tagged proteins. If you consider a three-step purification protocol, the step from IMAC to IEX can be carried out directly if the buffer and pH conditions chosen in IMAC are compatible. This can typically be achieved if anion exchange columns/resins are selected. Please keep in mind that imidazole is a buffer substance as well, and that a sufficient washing phase should be applied after sample loading.