Affinity Chromatography: Principle and Applications | Pharmacist Dunia

Understanding Affinity Chromatography: Principle and Applications

Affinity chromatography is a powerful method for purifying specific molecules from complex mixtures. Learn about the specific binding interactions between immobilized ligands and their binding partners, and the diverse applications of this technique in biological research.

 

Looking to purify specific molecules from complex mixtures? Learn about affinity chromatography - a powerful technique that uses immobilized ligands and specific binding interactions to purify molecules. Discover its diverse applications in biological research and the principle behind this highly specific separation technique.

 

Affinity chromatography is a highly specific separation technique used to purify molecules based on their specific binding interactions with immobilized ligands. This powerful method is widely used for the purification of a specific molecule or a group of molecules from complex biological mixtures. It relies on the principle of highly specific biological interactions between two molecules, such as interactions between an enzyme and its substrate, a receptor and its ligand, or an antibody and its antigen.

The diversity of affinity ligands and their potential targets has made affinity chromatography one of the most diverse and powerful chromatographic methods for biomolecule purification. It has a wide range of applications in various fields, including biochemistry, biotechnology, and medicine. Some common applications of affinity chromatography include the purification of enzymes, antibodies, antigens, hormones, and DNA-binding proteins.

 

If you are looking to purify and separate a desired protein using affinity chromatography, this article is a must-read. Learn about the specific and reversible interactions between protein and affinity ligands, and the essential components needed for successful separation. Discover how affinity support, spacer arms, and ligand work together for effective purification.

 

 

Affinity chromatography utilizes reversible interactions between a target protein and its affinity ligand to purify and separate large biomolecules from complex mixtures. A specific affinity ligand is selected based on the protein's inherent recognition site, and the binding must be specific and reversible.

 

When affinity chromatography is used for the purification and separation of large biomolecules from complex mixtures the following instruments or components are mandatory:

1.  Affinity Supports (Matrix)

2.  Spacer arms

3.  Ligands

 

Affinity Chromatography: Components and Ligands for Efficient Purification

Learn about affinity chromatography components and ligands for efficient purification. Discover the importance of affinity supports, spacer arms, and ligands.

1.  Affinity Supports (Matrix)

Traditionally, affinity chromatography uses porous support materials such as agarose, polymethacrylate, polyacrylamide, cellulose, and silica. These materials come in various particle and pore sizes, and some already have common affinity ligands immobilized, such as protein A, Cibacron Blue, and heparin.

•    Traditionally, affinity chromatography support materials have consisted of porous support materials such as agarose, polymethacrylate, polyacrylamide, cellulose, and silica.
•      All of these support materials are commercially available and come in a range of particle and pore sizes.
•     Some supports may be available with common affinity ligands already immobilized (e.g. protein A, Cibacron Blue, heparin).

2.  Spacer Arms

Spacer arms are critical in efficient binding and creating a better binding environment for the target molecule. Binding sites are often deeply located and difficult to access due to steric hindrance, so a spacer arm is incorporated between the matrix and ligand. The length of spacer arms is crucial, as too short or too long arms may lead to binding failure or non-specific binding. Generally, spacer arms are used when coupling molecules less than 1000 Da.

• Due to the fact that binding sites of the target molecule are sometimes deeply located and difficult to access due to steric hindrance, a spacer arm is often incorporated between the matrix and ligand.
•     It facilitates efficient binding and creates a more effective and better binding environment.
•    The length of these spacer arms is critical.
•    Too short or too long arms may lead to failure of binding or even non-specific binding.
•     In general, the spacer arms are used when coupling molecules less than 1000 Da.

3.  Ligands

Different types of compounds can be used as ligands in affinity chromatography, such as antibodies, dye-ligands, and DNA. Antibodies have high specificity and binding constants due to their variability in the amino acid sequence in the binding sites. Monoclonal antibodies are preferred in affinity chromatography because of their uniform affinity support. Dye-ligands are another type of affinity ligand that can be used to purify biomolecules from complex mixtures. DNA can also be used as an affinity ligand to purify DNA-binding proteins, DNA repair proteins, primases, helicases, polymerases, and restriction enzymes. The scope of biomolecules that can be purified using DNA is expanded when aptamers (single-stranded oligonucleotides) are utilized.

 

 

"Harnessing the power of different types of compounds as ligands is key in affinity chromatography. Antibodies offer high specificity and relatively large binding constants, making them ideal ligands. Dye-ligands are also effective in purifying biomolecules from complex mixtures. DNA is another affinity ligand that can purify a wide range of biomolecules, with aptamers expanding the scope even further." 

 

a) Antibodies as Affinity Ligands

·           Due to the variability of the amino acid sequence in the antibody binding sites they can be used as good ligands.

·           They have several advantages including their high specificity and relatively large binding constants.

·           Monoclonal antibodies are often more desirable than polyclonal antibodies in affinity chromatography due to their lack of variability which allows for the creation of a more uniform affinity support.

b) Dye-Ligands for Affinity Chromatography

·           Another type of affinity ligand that can be used to purify biomolecules from complex mixtures is a dye ligand.

·           The binding between the dye and enzyme caused this co-elution.

·           The dye-enzyme binding was utilized in the purification of pyruvate kinase using a Blue Dextran column in 1971.

c) DNA as an Effective Affinity Ligand

•     DNA can also be used as an affinity ligand.
•     It can be used to purify DNA-binding proteins, DNA repair proteins, primases, helicases, polymerases, and restriction enzymes.
•     The scope of biomolecules which can be purified using DNA is expanded when aptamers (single-stranded oligonucleotides) are utilized.

7 Applications of Affinity Chromatography for Protein Purification

Affinity chromatography is a powerful technique used for purifying specific biomolecules from complex mixtures. The technique relies on the specific interaction between the target biomolecule and a ligand that is immobilized onto a solid support.

 

There are many different ligands that can be used in affinity chromatography, including antibodies, dye ligands, and DNA. Each ligand has its own unique properties that make it suitable for purifying specific biomolecules.

 

Applications of affinity chromatography are numerous and varied, and include:

 

1.  Immunoglobulin Purification (Antibody Immobilization)

 

Antibodies can be immobilized by both covalent and adsorption methods. Random covalent immobilization methods generally link antibodies to the solid support via their free amine groups.

 

2.  Recombinant Tagged Proteins

 

Purification of proteins can be easier and simpler if the protein of interest is tagged with a known sequence commonly referred to as a tag. This tag can range from a short sequence of amino acids to entire domains or even whole proteins. Tags can act both as a marker for protein expression and to help facilitate protein purification.

 

3.  Protein A, G, and L Purification

 

Proteins A, G, and L are native or recombinant proteins of microbial origin which bind specifically to immunoglobulins including immunoglobulin G (IgG). These proteins can be purified using affinity chromatography.

 

4.  Biotin and Biotinylated Molecules Purification

 

If a biotin tag can be incorporated into a biomolecule, it can be used to purify the biomolecule using a streptavidin or avidin affinity support.

 

5.  Affinity Purification of Albumin and Macroglobulin Contamination

 

Affinity purification is a helpful tool for cleaning up and removing excess albumin and α2-macroglobulin contamination from samples using affinity chromatography.

 

 

6.  Lectin Affinity Chromatography

 

Lectin affinity chromatography is one of the most powerful techniques for studying glycosylation as a protein post-translational modification. Lectins are carbohydrate-binding proteins that can be used to purify glycoproteins from complex mixtures.

 

7.  Reversed Phase Chromatography

 

Reversed-phase chromatography is a kind of affinity interaction between a biomolecule dissolved in a solvent. Reversed-phase chromatography is generally more suitable for separating non-volatile molecules.

 

By utilizing the appropriate ligand and support, affinity chromatography can be a highly selective and effective method for purifying biomolecules for a wide range of applications in biotechnology, biochemistry, and pharmaceuticals.

 

 

In conclusion, affinity chromatography is a powerful method for the purification of specific molecules from complex biological mixtures. It is based on highly specific biological interactions between two molecules and has a wide range of applications in various fields of research. By understanding the principle and applications of affinity chromatography, researchers can take advantage of this powerful method for their specific research needs.