Advanced uses of IMAC Affinity Chromatography

Review Article

Austin Chromatogr. 2016; 3(1): 1042.

Advanced uses of IMAC Affinity Chromatography

Allen TakYiu Lo2,3, Tan HY2,3 and Bianco PR1,2,3*

¹Department of Biochemistry, University at Buffalo, USA

²Department of Microbiology and Immunology, University at Buffalo, USA

³Center for Single Molecule Biophysics, University at Buffalo, USA

*Corresponding author:Bianco PR, Center for Single Molecule Biophysics, Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY 14214, USA

Received: April 06, 2016; Accepted: May 18, 2016; Published: May 20, 2016


Ever since the development of molecular biology, various techniques have allowed researchers to engineer proteins of interest. Isolation of that protein from crude cell lysatesoften is the rate-limiting step in protein studies. Immobilized-Metal Affinity Chromatography (IMAC) provides one of the easiest methods for protein purification. It is a robust purification method resulting in nearly homogeneous protein (free of nucleases); which is suitable for any downstream characterization and studies such as crystallization or singlemolecule experiments. In this miniview, we provide insight to improve the protein homogeneity and summarize recent advanced uses of this method to isolate protein complexes formed in vivo.

Keywords: IMAC; Affinity chromatography; MBP


With the advances of cloning techniques, tagging proteins of interest has become a routine practice in the laboratory. There are different tags available to choose from for various purposes. Some tags are used for protein purification while others are used to track a protein in vivo. For example, fluorescent protein tags permit visualization of protein localization in the cell [1,2]. Some common tags used for purification purposes are Glutathione S-Transferees (GST) [3], Maltose-Binding Protein (MBP) [4], histidine (His) [5], and FLAG™ [6] etc. Histidine-tags (His-tag) are one of the most popular modifications used to facilitate protein purification. These tags consist of a stretch of four to ten consecutive histidine residues (with six being the most widely used [3]). They can be introduced at either the N- or C-terminus of the target protein to provide robust purification through an Immobilized Metal Affinity Column (IMAC).

This affinity column consists of a supporting matrix with an attached ligand and the immobilized metal ion. These are available from different manufacturers (Table 1). The most common supporting matrix consists of cross linked agarose beads (6%), which are large porous beads that provide high binding capacity of the resin while maintaining its stability in various pH and chemicals necessary during purification. Nitrilotriacetic Acid (NTA) is the most commonly used ligand to coordinate a metal ion on the column. It is a tetra dentate chelator that immobilizes the metal ion through four coordinate covalent bonds on the column. The remaining two coordination sites interact with the histidine side chains of the tag so that His-tagged protein can be retained on the column, which proteins lacking such a tag will flow through the column [3].