Affinity Chromatography Principles, Applications, and Techniques for Molecular Purification.pptx
0 likes995 views
Affinity chromatography is a technique for purifying molecules through specific interactions with ligands attached to a stationary phase, commonly used in biochemistry and pharmaceutical industries. The method relies on the reversible binding between the target molecule and the immobilized affinity ligand, allowing for selective isolation by altering mobile phase conditions. Various types of affinity chromatography, such as IMAC and antibody affinity chromatography, cater to different purification needs, including protein and antibody isolation.
More Related Content
Similar to Affinity Chromatography Principles, Applications, and Techniques for Molecular Purification.pptx (20)
Affinity Chromatography Principles, Applications, and Techniques for Molecular Purification.pptx
- 1. Affinity Chromatography: Principles, Applications, and Techniques for Molecular Purification Affinity Chromatography is a powerful technique used to purify and separate molecules based on specific interactions between the target molecule and a ligand attached to a stationary phase. It is widely applied in biochemistry, molecular biology, and pharmaceutical industries to isolate proteins, enzymes, antibodies, and nucleic acids. DR. SMITA KUMBHAR (M. Pharm. PhD), Associate Professor, Pharmaceutical Chemistry, HOD, Pharmaceutical Regulatory Affairs Department, Sanjivani College of Pharmaceutical Education and Research (Autonomous), Kopargaon, Maharashtra, India
- 2. Affinity Chromatography Affinity chromatography is a highly specific separation technique that leverages the strong and selective interactions between a target molecule (ligand) and a complementary binding partner (affinity ligand) immobilized on a stationary phase. This technique provides a powerful means of isolating and purifying a specific molecule from a complex mixture. The key to affinity chromatography lies in the specific and reversible interaction between the ligand and the affinity ligand. This interaction forms the basis for separating the desired molecule from other components in the mixture. Once the target molecule binds to the immobilized affinity ligand, it can be eluted by altering the conditions of the mobile phase, disrupting the interaction and releasing the purified molecule.
- 3. Affinity chromatography involves the covalent attachment of an immobilized biochemical (called an affinity ligand) to a solid support. When the sample is passed through the column, only the solute that selectively binds to the complementary ligand is retained. The other sample components elute without retention. The interaction between the ligand attached to the solid support of the stationary phase and the target molecule in the analyte is possible because of electrostatic, hydrophobic reaction, or hydrogen bonding. Nature of interaction between the target molecule and the ligand to help determine the selection of proper stationary phase and the mobile phase. The separation is well expressed by lock and key binding. The retained solute(s) can be eluted from the column by changing the mobile phase conditions.
- 7. Instrumentation for affinity chromatography Affinity chromatography involves using a column packed with a stationary phase, often a resin, to which an affinity ligand is attached. This ligand specifically binds to the target molecule, allowing it to be selectively isolated from the mixture. Instrumentation for affinity chromatography typically consists of a chromatography column, a pump to deliver the mobile phase, a detector to monitor the elution of the target molecule, and a fraction collector to collect the purified fractions. The choice of column depends on the scale of the purification and the flow rate required. The pump is essential for controlling the flow rate and pressure within the column. The detector, commonly a UV-Vis detector, monitors the absorbance of the eluent at specific wavelengths, indicating the presence of the target molecule. The fraction collector automatically collects eluted fractions, allowing for efficient isolation and analysis of the purified target molecule.
- 8. Stationary phase in Affinity Chromatography: The stationary phase is made of a solid matrix and ligand one more important component of the stationary phase in Affinity Chromatography. Component of stationary Phase in Affinity Chromatography. Ligand: Ligand is an immobilized chemically insoluble matrix supported by the stationary matrix. It reversibly adsorbs molecule species (affine components or target molecules) from a mixture of Analyte. Ligand is attached to the solid support. It exploits the unique property of extremely specific reversible biological interaction to achieve separation and purification. The separation exploits the “Lock n Key” binding that prevalent in a biological system. Ligands are coupled to affinity matrix are now commercially available and ready to use. Different types of ligand that bind to the substance which needs to purify: Antibody: This can be monoclonal or polyclonal. Highly specified and large binding constant DNA: Can be used for polymerase, DNA –binding proteins helicases, and restriction enzyme. Biometric dyes: used for protein Peptides: these are used for biomolecule
- 9. Spacer Arm: A spacer arm is the chain of carbon and/or other atoms that position a functional group away from the solid matrix to which it is covalently bound and makes it more available to a ligand and less restricted by steric hindrance by the matrix. Matrix: Solid Matrix or stationary phase is to provide the support to the ligand Solid matrix maybe porous or non-porous It should be of adequate size and shape i.e increase in particle size reduces the flow resistance and separation power & decrease in size leads to increase flow resistance and clogged. · Irregular shape leads to unequal path lengths for substance and band broadening. So spherical shape is best suited. · Mechanically and chemically stable and resistant against microorganisms. · It should be inert to prevent analyte from interacting non-specifically with matrix itself. · Economical · Eg. Agarose, Cellulose dextran Sepharose is a bead formed of agarose gel.
- 10. 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.
- 11. Types of affinity chromatography There are several types of affinity chromatography, each with its unique target and purification goals. 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 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.
- 12. Applications of Affinity Chromatography Protein Purification It is often used to purify enzymes, receptors, or antibodies. Antibody Isolation Affinity chromatography is ideal for isolating monoclonal or polyclonal antibodies from serum or cell culture media. Drug Discovery This method helps in identifying receptor-ligand interactions, making it useful in drug development.