Overview of Affinity Chromatography
Affinity chromatography involves the binding affinity between the target protein and a ligand immobilized on a stationary phase within a chromatography column.
One of the advantages of affinity chromatography is its high specificity and selectivity, which allows for the target protein’s purification to a high degree of purity. Additionally, affinity chromatography can be combined with other chromatography techniques, such as ion exchange chromatography or size exclusion chromatography, to purify the target protein further or separate it from other proteins in the sample.
This article will discuss affinity chromatography, its types, applications, and considerations. So, let’s first understand what affinity chromatography is in the next section.
Introduction to Affinity Chromatography
Summary: This section should go into more detail and define and describe affinity chromatography. This section should give readers a good understanding of when affinity chromatography is used, how it’s used, what conditions must be present, what it does, etc. It should also answer why this method is one of the most powerful.
Affinity chromatography is a powerful separation technique to purify and isolate specific biomolecules from a complex mixture. It is based on the principle of specific binding between a target molecule and an immobilized ligand in a stationary phase.
The stationary phase typically comprises a matrix, such as an agarose or Sepharose beads. The ligand can be an antibody, receptor, enzyme, or any other molecule that binds specifically to the target biomolecule.
The mobile phase is a buffer solution containing the complex biomolecule mixture passed through the affinity column. The target biomolecule binds to the immobilized ligand, while non-specific molecules pass through the column and are collected in the flow-through fraction.
The entire thing is summarized with the flow chart of the method provided below.
Affinity chromatography can be used for various biochemistry, molecular biology, and biotechnology applications. For instance, it can help purify proteins, nucleic acids, and other biomolecules for downstream analysis, such as mass spectrometry, western blotting, or DNA sequencing.
However, the success of affinity chromatography depends on several factors, such as the choice of immobilized ligand, mobile phase buffer, and elution conditions. That’s why optimizing the conditions to achieve maximum binding and elution of the target biomolecule while minimizing non-specific binding and impurities is important.
Now that we know affinity chromatography let’s look at how affinity purification works in detail in the next section.
How does affinity purification work?
Affinity purification uses an affinity column containing stationary and mobile/liquid phases. The stationary phase comprises a matrix, such as an agarose or Sepharose beads, which have been covalently linked to a ligand specific to the biomolecule of interest.
The mobile phase is a solution that contains a complex mixture of biomolecules, and it is passed through the column. Now, let’s discuss them in detail below.
Stationary phase
The stationary phase is an essential component of the affinity column. The matrix, such as agarose or Sepharose beads, provides a large surface area for the immobilization of the ligand. The ligand is covalently attached to the matrix through a stable chemical bond, such as an amide or thioether bond.
The ligand is selected based on its specific binding affinity for the target biomolecule. For instance, if the target biomolecule is a protein, the ligand could be an antibody or a receptor specific to that protein.
Mobile/Liquid phase
The mobile phase is a solution containing a complex mixture of biomolecules. This solution is passed through the column, and the target biomolecule is retained on the column due to its specific binding affinity for the immobilized ligand. The mobile phase can be a buffer, a cell lysate, or a tissue extract, depending on the application.
The biomolecules that do not have an affinity for the immobilized ligand will pass through the column and be collected in the flow-through fraction. The target biomolecule is then eluted from the column using a specific elution buffer that disrupts the binding interaction between the ligand and the biomolecule.
Affinity purification exploits the binding affinity between a biomolecule and an immobilized ligand on a stationary phase. The biomolecule is isolated and purified from a complex mixture of biomolecules in the mobile phase. This technique is widely used in biochemistry, molecular biology, and biotechnology to isolate and purify proteins, nucleic acids, and other biomolecules.
Types of affinity chromatography
There are several types of affinity chromatography, each with its unique target and purification goals. So, let’s look at them one by one in detail.
Immobilized Metal Ion Affinity Chromatography (IMAC)
IMAC is a common method used to purify His-tagged recombinant proteins. The His-tag binds to immobilized metal ions, usually Ni2+ or Co2+, coordinated with chelating agents such as iminodiacetic acid or nitrilotriacetic acid. The target protein is then eluted by adding an excess of imidazole or histidine to compete with the His-tag for binding to the metal ion.
Antibody Affinity Chromatography
It is a common chromatography method used for purifying proteins with high specificity and affinity for antibodies. Antibodies can be immobilized on a solid support, such as Sepharose or agarose, and then used to capture the target protein. The target protein is then eluted by changing the pH or by using an excess of the antigen.
Lectin Affinity Chromatography
This affinity chromatography type helps purify glycoproteins with specific sugar moieties that can bind to lectins. Lectins are immobilized on a solid support and help capture the glycoprotein. The target protein is eluted by an excess specific sugar for the lectin protein to bind.
Protein A/G Affinity Chromatography
Protein A/G affinity chromatography is commonly used to purify immunoglobulins (IgGs) from serum or other biological fluids. Proteins A and G bind to the Fc region of IgGs and are immobilized on a solid support, such as Sepharose or agarose. The target protein is eluted by changing the pH or using excess Fc-specific peptides.
Dye Ligand Affinity Chromatography
Finally, dye ligand affinity chromatography allows the purification of proteins with exposed hydrophobic regions. Various dye ligands, such as Cibacron Blue, are immobilized on a solid support and help capture the target protein. The target protein is then eluted with high salt or a competitive hydrophobic ligand. Besides these, other types of affinity chromatography are used for the purification process. These are described in detail below.
Applications of affinity tags
Affinity chromatography is a powerful tool for purifying proteins, and one of the most common applications of affinity chromatography is using affinity tags. Affinity tags are short peptide sequences fused to the target protein and selectively bind the protein to a stationary phase in the chromatography column. Here are a few examples of the various applications of affinity tags:
Purification of Recombinant Proteins
Affinity tags help purify recombinant proteins engineered to contain the tag. The tag allows the protein to be selectively bound to a stationary phase in the chromatography column, simplifying the protein purification from other cellular components.
Protein-Protein Interaction Studies
Affinity tags can help pull down interacting proteins from a complex mixture. For instance, one protein can be fused to an affinity tag to pass the mixture through a chromatography column containing the stationary phase that specifically binds the tag. The interacting protein will be co-purified with the tagged protein, allowing for the identification of interacting partners.
Protein Localization Studies
Affinity tags can also help visualize the protein localization within a cell. Fluorescent tags are attached to the affinity tag, so the proteins are visualized by fluorescence microscopy.
Drug Discovery
Affinity tags are beneficial in drug discovery to screen for compounds that selectively bind to a target protein. The protein can be immobilized on a stationary phase using the affinity tag, and compounds can be screened for their ability to bind to the protein.
Considerations for affinity purification
There are several factors to consider when performing affinity purification. These are listed below:
- The selection of the immobilized ligand is vital to determine the purification’s specificity and efficiency.
- The choice of mobile phase and buffer conditions can affect the binding of the target biomolecule to the immobilized ligand.
- Optimizing the elution conditions is essential to ensure the maximum yield and purity of the target biomolecule. Additionally, the cost and availability of the ligand and the stationary phase also play an important role and must be considered.
However, there are also some limitations to affinity purification. These are listed below:
- The target biomolecule must have a high affinity for the immobilized ligand. If the affinity is too low, the purification will be inefficient.
- The target biomolecule may be present in very low abundance, making it difficult to isolate and purify.
Note: The conditions for affinity purification can vary depending on the application, and careful optimization is necessary to achieve optimal results.
Other types of chromatography for purification
There are other types of chromatography used for purification in specific situations. Here are a few examples:
Hydrophobic Interaction Chromatography (HIC)
This technique is useful for separating proteins based on their hydrophobicity. The stationary phase in HIC columns contains hydrophobic ligands and proteins with different hydrophobic properties that interact differently with the stationary phase.
Reversed Phase Chromatography (RPC)
This technique is similar to HIC but uses a hydrophobic stationary phase and a polar mobile phase. In this chromatography type, proteins with different hydrophobicity interact differently with the stationary phase and are separated based on their polarity.
Immobilized Metal Affinity Chromatography (IMAC)
This technique is similar to affinity chromatography, but the stationary phase contains metal ions that bind to histidine residues on the protein. This technique can help purify the recombinant proteins that contain a histidine tag.
Multi-Angle Light Scattering (MALS) Chromatography
Light scattering is used to measure proteins' size and molecular weight as they pass through a chromatography column. It can help determine the oligomeric state of proteins and to detect protein aggregates.
Learn more about chromatography
Chromatography is a powerful tool for separating and purifying biomolecules. To learn more about chromatography and how to select the right chromatography resin, you can refer to the resources from Avantor Sciences.
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