Affinity Chromatography, principal, process, application.pptx

Affinity Chromatography, principal, process, application

1. AFFINITY CHROMATOGRAPHY SUBMITTED BY PRIYANSHU SETH ROLL NO – 343 SESSION-(22-25, 6 TH SEMESTER)

2. CONTENTS OF TECHNIQUE

3. PRINCIPAL OF AFFINITY CHROMATOGRAPHY 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 . It is based on highly specific biological interactions between two molecules such as interactions between enzyme and substrate , receptor and ligand , or antibody and antigen. These interactions which are typically reversible are used for purification by placing one of the interacting molecules referred to as affinity ligand onto a solid matrix to create a stationary phase while a target molecule is in the mobile phase . Many of the commonly used ligands coupled to affinity matrices are now commercially available and are ready to use.

4. An ideal chromatographic bed material (matrix) It must possess suitable groups to which ligand can be covalently coupled. Many groups may be introduced into matrix to couple ligands. They may be nucleo philic as NH2, SH, OH or electrophilic such as activated acid chlorides, carbonyls activated by carbodiimide, iothiocyanate or diazonium salts. It must remain unchanged under the conditions of attachment of ligand. During the binding of the macromolecule and its subsequent displacement from ligand, it must retain its physical and chemical stability . It must not exhibit non-specific adsorption. It should have an open pore structure. The most commonly used matrices are cross-linked dextrans (e.g. Sephacryl ), agarose (e.g. Sepharose), polyacrylamide gel (Bio gel P), polystyrene, cellulose, porous glass and silica .

5. How Ligand Binds with Matrix? For attachment of the ligand with the matrix, the matrix is given preliminary treatment with cyanogens bromideat pH11. This causes activation of the matrix and the molecules containing primary amino groups could then easily be coupled to CNBr activated matrices . Different spacer arms including 1,6-diamino hexane, 6-amino hexanoic acid, and 1,4-bis(epoxy- propoxy )butane have been used to which the ligand can be attached by conventional organo synthetic procedures involving the use of succinic anhydride and a water soluble carbodiimide . A number of supports of agarose, dextran and polyacrylamide type are commercially available with a variety of spacer arms and ligands.

6. Ligand Design

7. COMPONENTS OF AFFINITY CHROMATOGRAPHY Stationary Phase (Matrix): A solid support where ligands are immobilized . Common materials include agarose, cellulose, and silica beads . Ligand: A specific molecule that binds the target biomolecule. Examples include antibodies, protein A/G, metal ions, lectins, and DNA/RNA sequences. Mobile Phase (Buffers): Binding Buffer – Promotes interaction between ligand and target. Washing Buffer – Removes unbound impurities. Elution Buffer – Releases the target molecule (pH change, salt gradient, or competitive binding). Chromatography Column: Holds the stationary phase and ensures controlled flow of the sample and buffers. Can be pre-packed or manually filled . Detection System: Confirms the purity of the eluted target using UV spectrophotometry, SDS-PAGE, or Western blotting .

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9. TYPE OF AFFINITY CHROMATOGRAPHY 1️⃣ Immunoaffinity Chromatography (IAC): Uses antibodies to capture specific antigens or proteins (e.g., antibody purification). 2️⃣ Metal Chelate Affinity Chromatography (IMAC): Utilizes metal ions (Ni²⁺, Co²⁺, Zn²⁺, Cu²⁺) to bind His-tagged proteins . 3️⃣ Lectin Affinity Chromatography: Uses lectins to bind glycoproteins and carbohydrate-containing molecules. 4️⃣ Protein A/G Affinity Chromatography: Specifically purifies immunoglobulins (antibodies) using Protein A or G . 5️⃣ Dye-Ligand Affinity Chromatography: Employs synthetic dye ligands to bind enzymes and proteins based on structural similarities. 6️⃣ Nucleic Acid Affinity Chromatography: Uses DNA or RNA sequences to purify nucleic acid-binding proteins . 7️⃣ Heparin Affinity Chromatography: Binds coagulation factors, growth factors, and plasma proteins using heparin as a ligand.

10. EXPERIMENTAL PROCEDURE

11. APPLICATIONS Application Examples Description Protein Purification Enzymes, antibodies, recombinant proteins Used for purifying proteins based on specific interactions with ligands. Nucleic Acid Isolation DNA, RNA Isolates specific DNA or RNA sequences using complementary nucleotide sequences as ligands. Antibody Purification Monoclonal and polyclonal antibodies Purifies antibodies using ligands such as protein A, protein G, or protein L. Enzyme Purification Specific enzymes Isolates enzymes using substrate analogs or inhibitors as ligands. Biopharmaceutical Production Monoclonal antibodies, insulin, therapeutic proteins Ensures high purity and activity of biopharmaceuticals for clinical use. Drug Development Screening potential drug candidates Identifies and screens drug candidates based on binding interactions with target molecules.

12. APPLICATIONS Food Additive Purification Enzymes, Flavors, food additives Purifies food additives to ensure product quality and safety. Quality Control Detecting contaminants in food products Used in quality control to detect and quantify specific contaminants or adulterants in food products. Protein-Protein Interactions Studying complexes between target proteins and binding partners Studies interactions between proteins by isolating complexes formed between them. Protein-DNA Interactions Understanding gene regulation and transcriptional control Studies interactions between proteins and DNA, important for understanding gene regulation. Biomarker Discovery Identifying biomarkers for disease diagnosis and prognosis Used in discovering and validating biomarkers for diagnostics. Diagnostic Assays Enzyme-linked immunosorbent assays (ELISAs) Develops diagnostic assays by capturing and detecting target antigens using specific antibodies. Pollutant Detection Detecting heavy metals and organic contaminants in the environment Detects and quantifies environmental pollutants using specific ligands. Bioremediation Isolating enzymes or microorganisms for degrading or detoxifying environmental pollutants Used in bioremediation processes to purify enzymes or microorganisms for pollutant degradation. Application Examples Description

13. ADVANTAGES AND DISADVANTAGES Advantages High Specificity : Highly selective purification based on specific interactions. Efficiency : Can purify complex mixtures in a single step. High Purity : Provides high levels of purity, suitable for sensitive applications. Versatility : Can be used to purify a wide range of biomolecules. Scalability : Can be adapted for small-scale and large-scale applications. Automation Compatibility : Suitable for high-throughput applications and reduces manual labor. Disadvantages Cost : Expensive ligands and matrices, especially for large-scale applications. Ligand Leakage : Risk of ligand leaking from the matrix, contaminating the purified product. Limited Reusability : Columns and matrices may degrade over time. Complex Sample Preparation : Time-consuming optimization of binding and elution conditions. Scalability Issues : Challenges in scaling up for industrial production. Potential for Non-Specific Binding : Risk of non-specific binding reducing purity.

14. CONCLUTION Affinity chromatography is a powerful and versatile technique for biomolecule purification. It relies on specific interactions between the target molecule and a ligand. The technique offers high specificity, efficiency, and purity. Applications include biotechnology, pharmaceuticals, food industry, and research. Challenges include cost, ligand leakage, and scalability issues. Ongoing innovations are improving the technique, such as stable ligands, automation, and miniaturization. Affinity chromatography continues to be relevant and significant in scientific research and industrial applications.