Immobilized+Metal+Affinity+Chromatography+(IMAC)

Htet San = Immobilized Metal Affinity Chromatography  =



Description :
Immobilized Metal Affinity Chromatography (IMAC) is a method used to separate and purify the proteins acco rding to their affinity to the column. This specific type of chromatography works by allowing the covalent bonding between the amino acid, histidine, in the protein and the metals ions that are immobilized in the column.

**Procedure: **

Purification of One Target Protein:
The stationary solid phase of the column was prepared by mixing the agarose beads, a metal of interest, and the metal-chelating agents, such as nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA). As outlined in Figure 2, the metal will bind to the metal-chelateing arm that is attached to the gel beads. This resultant resin is then packed into a column to begin the chromatography. In proteins, histidine is a particular amino acid that is found to have a high affinity for metal ions. When the protein is added into the column, its polyhistidine tag (6XHis) on the N- or C-terminus binds with the metal-chelating arm. Then, increasing concentration of imidazole is added to the column to wash out the unspecific binding of proteins. Proteins that have a lower binding affinity to the metal chelator will be eluted first as the imidazole replaces with the protein. As more concentration of imidazole added, the proteins with the highest affinity to the metal ion will be eluted last. The success of the purification is later analyzed by running the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Analysis of Protein-protein Interaction (The Pull-Down Assay):
Immobilized metal affinity chromatography can also be used to analyze the binding affinity of two proteins. After washing out the unbound proteins in the column, a secondary protein is added to the tagged protein. The tagged protein becomes the "bait," and the protein whose binding affinity is in question is known as the "prey." If the two proteins have strong interactions with one another, the prey protein will pull down the bait protein, eluting both proteins out in the wash. If the prey protein has a higher affinity to the column, only the bait protein will be eluted out in the final wash. The protein-protein interaction can be analyzed by running SDS-PAGE analysis of the eluted fraction. Presence of two bands in relatively equal concentration indicates that the proteins have a high affinity towards each other.



Origin:
Since the early 1900's, scientists had observed the specificity of certain enzymes that can bind to certain molecules (Scouten, 1991). However, it was not until 1910 that Emil Starkenstein developed the first form of affinity chromatography to purify alpha-amylase enzyme using its affinity to the starch molecules. After Starkenstein, many other scientists began to adopt the idea of using molecular affinity for purification. But they had only yielded incomplete purification and found that the procedure was extremely laborious. It wasn't until Pedro Cuatrecasas' experiment, involving affinity chromatography that the technique gained its credibility in the scientific community (Cuatrecasas, 1968). The use of affinity chromatography has become more accurate and faster as the scientists gained the ability to learn about the structure of the molecules they want to purify. The technique has now developed to purify the biochemical mixture based on the interaction, such as between metals and chelate buffers.

Experiments:
Some notable experiments using immobilized metal affinity chromatography include identifying and characterizing the recombinant proteins in Escherichia coli (Tiwari, 2010). Influenza virus A/PuertoRico/8/34 was also purified from a cell culture using this inexpensive and rapid method (Opitz, 2009). Advances are still being made to the technique. One study integrated IMAC with preparative electrochromatography to study the intensification effects of electric field on the technique when it is used for protein purification (Shi, 2011).

** Recent Research: **

In this publication by the students and faculties from SUNY Oswego, metal binding properties of the human serum proteins under physiological conditions were analyzed using the metal affinity chromatography. While the binding of protein to the metal play an important role in maintaining homeostasis, excess of metals in the blood can cause diseases and health problems. Knowing the protein-metal binding affinity can open up the potential targets for studies of effects that the metals have on the human body (Wang et al., 2013).

Research done on marine cyanobacteria revealed that they have a mechanism to uptake zinc utilizing their protein metal binding affinity. Scientists tested the protein-metal binding affinity of porin from the cyanobacteria using IMAC loaded with zinc. They found that there are surface exposed sites in the porin to uptake zinc in the organism. Not only can this research on the binding of zinc to the proteins in the Gram-negatvie bacteria can explore the survival of these bacteria in the environment with low concentration of zinc, it also gave a new perspective on the relationship between mammalian host to infections (Barnett et al., 2014).

In one of the agricultural studies, the researchers grew rice in the copper stressed environment, and the rice root samples were run down the IMAC column tagged with copper to purify the proteins that are involved in the growth of the rice plants. It was found that the proteins necessary in the antioxidant defense, carbohydrate metabolism, nucleic acid metabolism, protein folding and stabilization, protein transport and cell wall synthesis of rice were identified after the eluted sample was purified (Song et al., 2014).

References:
Barnett, J.P., Scanlan, D.J., & Blindauer, C.A. (2014). Identification of major zinc-binding proteins from a marine cyanobacterium: insight into metal uptake in oligotrophic environments. //The Royal Society of Chemistry, 6.// doi: 10.1039/C4MT00048J.

Block, H., Maertens, B., Spriestersbach, A., Brinker, N., Kubicek, J., Fabis, R....Schäfer, F. (2009). Chapter 27 Immobilized-Metal Affinity Chromatography (IMAC): A Review. //Methods in Enzymology,// //463,// 439-473. doi: 10.1016/S0076-6879(09)63027-5.

Cuatrecasas, P., Wilchek, M., & Ansifen, C. B. (1968). Selective enzyme purification by affinity chromatography. //Proceedings of National Academy of Sciences of the U.S. 61,// 636-643.

Hage, D.S., & Cazes, J. (2006). Handbook of affinity chromatography (2nd ed.). Boca Raton, FL: Taylor & Francis Group.

Opitz L., Hohlweg J., Reichl U., Wolff M.W. (2009). Purification of cell culture-derived influenza virus A/Puerto Rico/8/34 by membrane-based immobilized metal affinity chromatography Journal of Virological Methods, 161 (2), pp. 312-316.

<span style="font-family: Webdings,sans-serif;">Scouten, W.H. (1991). Affinity Chromatography for Protein Isolation. Current Opinion in Biotechnology. 2(1): 37-43

<span style="font-family: Webdings,sans-serif;">Shi, Q., Jia, G. & Sun, Y. (2011). Process intensification of immobilized metal affinity chromatography with longitudinal and oscillatory transverse electric fields. Separation and Purification Technology, 77(3): 375-381

Song, Y., Zhang, H., Chen, C., Wang, G., Zhuang, K., Cui, J., & Shen, Z. (2014) Proteomic analysis of copper-binding proteins in excess copper-stressed rice roots by immobilized metal affinity chromatography and two-dimentsional electrophoresis. //BioMetals, 27(2),// Retrieved from http://link.springer.com/article/10.1007/s10534-014-9707-x.

<span style="font-family: Webdings,sans-serif;">Tiwari N., Woods L., Haley R., Kight A., Goforth R., Clark K....Beitle R. (2010). Identification and characterization of native proteins of Escherichia coli BL- 21 that display affinity towards Immobilized Metal Affinity Chromatography and Hydrophobic Interaction Chromatography Matrices. Protein Expression and Purification, 70(2): 191-195.

<span style="font-family: Webdings,sans-serif;">Wang, F., Chmil, C., Pierce, F.,Ganapathy, K., Gump, B.B., MacKenzie, J.A… Bendinskas, K. (2013). Immobilized metal affinity chromatography and human serum proteomics. //Journal of Chromatography B//, 934.