Protein purification by immobilized metal affinity chromatography (IMAC) using a VIAFLO 96/384 handheld electronic pipette (2023)

FAQs

What is immobilized metal affinity chromatography IMAC? ›

Immobilized metal affinity chromatography (IMAC) is a powerful protein fractionation method used to enrich metal-associated proteins and peptides. In proteomics, IMAC has been widely employed as a prefractionation method to increase the resolution in protein separation.

Some recombinant proteins are engineered to have two hexahistidine tags. His-tag purification uses the purification technique of immobilized metal affinity chromatography, or IMAC. In this technique, transition metal ions are immobilized on a resin matrix using a chelating agent such as iminodiacetic acid.

Immobilized metal affinity chromatography (IMAC) is the most widely used method to purify the proteins according to their affinity to specific metal ions, which was first introduced by Porath (1989). This involves the use of phosphate affinity metals which are chelated on resin or beads and packed in a column.

The metals are immobilized onto a chromatographic medium by chelation. Certain amino acids, e.g. histidine and cysteine, form complexes with the chelated metals around neutral pH (pH 6–8) and it is primarily the histidine-content of a protein which is responsible for its binding to a chelated metal.

The IMAC principle is briefly constituted of four main steps (Fig. 19.10): (1) metal-ion immobilization onto the matrix, (2) Sample injection and target protein/peptide retention, (3) washing step to eliminate the unbound components, and (4) finally, elution of the target biomolecule.

The principle of IMAC is based on the interaction between metal ions immobilized on the chromatographic support material and the lone pair electrons of nitrogen-or oxygen-containing amino side chains (see Fig. 3.12a).

The most widely used elution buffer for affinity purification based on protein interactions is 0.1 M glycine•HCl, pH 2.5-3.0. This buffer effectively dissociates most protein:protein and antibody:antigen binding interactions without permanently affecting protein structure.

The principle of affinity chromatography is that the stationary phase consists of a support medium (e.g. cellulose beads) on which the substrate (or sometimes a coenzyme) has been bound covalently, in such a way that the reactive groups that are essential for enzyme binding are exposed.

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The most widely used method for protein purification is affinity chromatography, which separates proteins based on their specific interaction with a matrix. It is one of the most effective techniques, since it takes advantage of the incorporation of a structure of choice (called a tag) onto the protein.

How does protein purification work? ›

Immunoaffinity chromatography uses the specific binding of an antibody-antigen to selectively purify the target protein. The procedure involves immobilizing a protein to a solid substrate (e.g. a porous bead or a membrane), which then selectively binds the target, while everything else flows through.

Protein A chromatography is the most frequently used affinity chromatography method in biomanufacturing. It is the standard technique for capturing recombinant monoclonal antibodies, which relies on the reversible and specific binding between the immobilized protein A ligand and antibodies.

Affinity chromatography separates proteins on the basis of an interaction between a protein and a specific ligand. The binding of the protein to a ligand attached to a matrix is reversed by either competition or by decreasing the affinity with pH and/or ionic strength.

Affinity Chromatography Steps

Competing molecules bind to the ligand, displacing the protein of interest. This competing molecule is typically removed from the protein of interest either through another chromatographic procedure or dialysis.

Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits.

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Elution and recovery of captured His-tagged protein from an IMAC column is accomplished by using a high concentration of imidazole (at least 200 mM), low pH (e.g., 0.1 M glycine-HCl, pH 2.5) or an excess of strong chelators (e.g., EDTA). Imidazole is the most common elution agent.

Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography.

How does chromatography work simplified? ›

How Does Chromatography Work? Chromatography is based on the principle where molecules in a mixture are smeared onto a solid or surface, and a stable phase (fluid stationary phase) separates the components of a mixture from each other while working with the aid of a mobile phase.

If a buffer containing more than one protein is used with an anion exchange resin, then the most negatively-charged protein will be most attracted to the stationary phase and will therefore elute last and the protein with the highest positive charge will elute first.

Why Use Affinity Chromatography? Affinity chromatography offers high selectivity, resolution, and capacity in most protein purification schemes. It has the advantage of utilizing a protein's biological structure or function for purification.

The molecules that are larger than the pore volume will be eluted first due to their inability to penetrate the pores. Whereas the smaller molecules will take a longer time to be eluted because they can penetrate into the pores (Fig. 6.6).

Unlike other purification methods such as gel filtration and size-exclusion chromatography, affinity chromatography manipulates specific molecular properties and binding interactions between molecules to purify the protein of interest.

Affinity chromatography is one of the most diverse and powerful chromatographic methods for purification of a specific molecule or a group of molecules from complex mixtures.

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What are the basic steps of protein purification? ›

There are four basic steps of protein purification: 1) cell lysis, 2) protein binding to a matrix, 3) washing and 4) elution.

For proteins, it is possible to use the following techniques either in a single step or sequentially: hydrophobic interaction column chromatography, size exclusion chromatography, ion exchange column chromatography, and affinity chromatography.

Purified proteins often need to be stored for an extended period of time while retaining their original structural integrity and/or activity. The extent of storage 'shelf life' can vary from a few days to more than a year and is dependent on the nature of the protein and the storage conditions used.

Conclusion

Protein purification involves extraction and purification. In extraction process, there are different procedures to disrupt cells or tissues as well as different extraction solvents, depending on the nature of the cells or tissues.

Membrane proteins are difficult to purify because they are present in low levels and they require detergents to become soluble in an aqueous solution. The selection of detergents suitable for the solubilization and purification of a specific membrane protein is critical in the purification of membrane proteins.

Phenol-Chloroform Extraction

Phenol chloroform extraction, normally followed by ethanol precipitation, is the traditional method to remove protein from a DNA sample. In this procedure, the DNA solution is mixed with phenol and chloroform.

Affinity chromatography (AC) exploits specific interactions among molecules, for example, the binding of a protein to its in vivo binding partner or an antibody. Affinity chromatography is widely used for protein purification.

It is a powerful tool to remove process-related impurities such as host cell proteins, DNA, endotoxin and leached Protein A, product-related impurities such as dimer/aggregate, endogenous retrovirus and adventitious viruses such as parvovirus, pseudorabies virus.

Performing a chromatographic experiment is basically a three-step process: 1) application of the sample, 2) "developing" the chromatogram by allowing the mobile phase to move up the paper, and 3) calculating Rf values and making conclusions. The distance traveled by the spot is measured to the MIDDLE of the spot.

What is the purpose of Affinity Chromatography? To separate and purify a single protein from solution using a ligand for the target protein attached to a matrix.

What is the importance of chromatography in protein analysis? ›

In any proteomic analysis, the first and most important task is the separation of a complex protein mixture, i.e. the proteome. Chromatography, one of the most powerful methods of separation, employs one or more inherent characteristics of a protein-its mass, isoelectric point, hydrophobicity or biospecificity.

Chromatography is one of the most common methods for the purification of recombinant proteins, and more specifically affinity chromatography is the one that is mostly used due to its high specificity, which allows us to obtain great purity in one single step.

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.

Nickel Columns for Chromatography. Nickel columns are used for immobilized metal affinity chromatography (IMAC) for the purification of recombinant proteins with a polyhistidine tag on either terminus. The most common tag is a hexahistidine tag (6xHis tag or His6 tag).

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Affinity chromatography is a method for selective purification of a molecule or group of molecules from complex mixtures based on highly specific biological interaction between the two molecules.

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Elution and recovery of captured His-tagged protein from an IMAC column is accomplished by using a high concentration of imidazole (at least 200 mM), low pH (e.g., 0.1 M glycine-HCl, pH 2.5) or an excess of strong chelators (e.g., EDTA). Imidazole is the most common elution agent.

Agarose gel electrophoresis is a method used to separate DNA molecule(s) on the basis of their molecular weight. Stained agarose gels can be used to estimate the molecular weight and purity of the DNA fragment(s) in a given sample. The conformation of a DNA molecule can affect its migration through an agarose gel.

There are four basic steps of protein purification: 1) cell lysis, 2) protein binding to a matrix, 3) washing and 4) elution.

How do you purify protein with his tag? ›

His-tagged proteins can be purified by a single-step affinity chromatography, namely immobilized metal ion affinity chromatography (IMAC), which is commercially available in different kinds of formats, Ni-NTA matrices being the most widely used.

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