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Affinity Chromatography.
The document is a seminar presentation on affinity chromatography presented by Ms. T. Jayasree under the guidance of Dr. P. Ramalingam at Raghavendra Institute of Pharmaceutical Education and Research. It explains the principles, procedure, and applications of affinity chromatography as a purification technique for biological molecules, highlighting its specificity in binding target proteins to immobilized ligands on a stationary phase. Various types of ligands, solid matrices, and methods for conducting the chromatography process are discussed, as well as its importance in genetic engineering and vaccine production.
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Affinity Chromatography.
- 1. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1 A Seminar as a part of curricular requirement for I year M.Pharm I Semester Presented by Ms. T.Jayasree (20L81S0709) Pharmaceutical analysis Under the guidance/Mentorship of Dr.P.Ramalingam., M.Pharm, Ph.D Director- R&D Division, Professor of pharmaceutical analysis and medicinal chemistry President - IPA local branch - Anantapuramu Affinity Chromatography
- 2. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2 • Introduction • Principle • Stationary Phase • Mobile phase • Affinity Media • Chromatographic Media • Immobilized ligands • Attachment of ligands to the matrix • Procedure • Batch and column setup • Applications Contents
- 3. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3 • Affinity chromatography was discovered by Pedro Cuatrecasas and Meir Wilcheck. • Affinity Chromatography is essentially a sample purification technique , used primarily for biological molecules such as proteins. • It is a method of separating a mixture of proteins or nucleic acids by specific interactions of those molecules with a component known as ligands , which is immobilized on a support. Introduction
- 4. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4 • If a solution of say , a mixture of proteins is passed over the column , one of the proteins bind to the ligand on the basis of specifity and high affinity. • Ex:- Enzymes for their substrate Antibodies for their antigens
- 5. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5 • The Principle 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. Principle
- 6. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
- 7. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7 • It is an insoluble solid matrix (porous or non-porous) covalently bonded to an immobilized ligand separated by a spacer arm Solid matrix (support material): • Natural : cellulose,agarose,dextrose • Synthetic : polymethacrylate ,polystyrene, polyacrylamide etc. • Inorganic : porous silica ,glass etc. Stationary phase
- 8. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8 Spacer arm: • Creates spaces between matrix and ligand • Provide better interaction between the ligand and target molecule by overcoming steric hindrance Affinity ligand: • It is a molecule that binds to specific target molecule reversibly .
- 9. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9 • Mobile phase – buffer solution Binding buffer ( equilibrium and sample application): Optimize the conditions to ensure the binding of target molecules to the specific ligand . Elution buffer (Elution) – Conditions are reversed to weaken the interaction between the target molecules and ligand to elute the target molecules. Wash buffer (Washing) - Conditions suitable to wash the unbound molecule without eluting the target molecules. Mobile phase
- 10. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10 • Amino acids – serum proteins, proteins, peptides, enzymes as well as rRNA dsDNA. • Avdin – Biotin – purification process of avidin , biotin and their derivatives . • Carbohydrates – glycoprotein's carbohydrate containing substance lectins . • Dye ligands : biological substrates and proteins . • Glutathione – GST tagged recombinant proteins . • Heparin – plasma coagulation proteins , nucleic acid enzymes and lipases Affinity Media
- 11. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11 • A matrix in its use here is a substance, usually in bead form to which a specific ligand is covalently bound. • In order for the matrix to be effective it must have certain characters: • It must be insoluble in solvents and buffers employed in the process. • It must be chemically and Mechanically stable. Chromatographic Media
- 12. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12 • It must be easily coupled to a Ligand or Spacer arm onto which the ligand can be attached. • It must exhibit good flow properties and have a relatively large surface area for attachment.
- 13. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13 • The Ligand can be selected only after the nature of the macromolecule to be isolated is known . • When a hormone receptor protein is to be purified by affinity chromatography, the hormone itself is an ideal candidate for the ligand . • For antibody isolation, an antigen or hapten may be used as a ligand. • If an enzyme is to be purified, a substrate analog , inhibitor, cofactor , or effectors may be used as an immobilized ligand . Immobilized Ligand
- 14. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14 • Several procedures have been developed for the covalent attachment of the ligand to the stationary phase. All procedures for gel modifications proceed in two separate chemical steps. • Activation of the functional groups on the matrix . • Joining of the ligand to the functional groups on the matrix. Attachment of Ligand to Matrix
- 15. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15 1. Affinity medium is equilibrated in binding buffer. 2. Sample is applied under conditions that favor specific binding of the target molecule(s) to a complementatary binding substance (the ligand),Target substances bind specifically, but reversibily,to the ligand and unbound material washes through the column. 3. Target protein is recovered by changing conditions to favor elution of the bound molecules, Elution is performed specifically, using a competitive ligand, or non- specifically ,by changing the pH,ionic strength or polarity ,Target protein is collected in a purified, concentrated form. Procedure
- 16. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16 • 4.Affinity medium is re-equilibrated with binding buffer.
- 17. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17 • Binding to the solid phase may be achieved by column chromatography whereby the solid medium is packed onto a column , the intial mixture run through the column to allow setting , a wash buffer run through the column and the elution buffer subsequently applied to the column and collected. • BATCH AND COLUMN SETUP
- 18. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18 • These steps are usually done at ambient pressure . • Alternatively, binding may be achieved using a batch treatment, for example, by adding the intial mixture to the solid phase in a vessel, mixing, seperatingthe solid phase, removing the liquid phase,washing, re-centrifuging, adding the elution buffer, re- centrifuging and removing the eluate.
- 19. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19 • Sometimes a hybrid method is employed such that the binding is done by the batch method, such that the binding is done by the batch method, but the solid phase with the target molecule bound is packed onto a column and washing and elution are done on the column. • A third method, expanded bed adsorption,which combines the advantages of the two methods mentioned above,has also been developed.
- 20. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20 • The solid phase particles are placed in a column where liquid phase is pumped in form the bottom and exits at the top. • The gravity of the particles ensure that the solid phase does not exit the column with the liquid phase . • Affinity columns can be eluted by changing salt concentrations,pH,pl,charge and ionic strength directly or through a gradient to resolve the particles of interest .
- 21. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21 Column setup Batch setup Steps
- 22. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 22 1. It is used for isolation and purification of all biological macromolecules. 2. It is used to purify nucleic acid , antibodies , and enzymes . 3. To notice which biological compounds bind to a particular substance . 4. To reduce an amount of substance in a mixture. 5. Used in Genetic Engineering-nucleic acid purification. 6. Production of Vaccines-antibody purification from blood serum. 7. And Basic Metabolic Research-protein or enzyme purification from cell free extracts. Applications
- 23. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 23 1. Parikh I, Cuatrecasas P. Affinity chromatography, principles and applications. InMethods of Protein Separation 1975 (pp. 255-276). Springer, Boston, MA. 2. Boyer R. Modern experimental biochemistry. Pearson Education India; 2000. 3. Sawhney SK, Singh R, editors. Introductory practical biochemistry. Alpha Science Int'l Ltd.; 2000. 4. Noctor TA, Wainer IW, Hage DS. Allosteric and competitive displacement of drugs from human serum albumin by octanoic acid, as revealed by high-performance liquid affinity chromatography, on a human serum albumin-based stationary phase. Journal of Chromatography B: Biomedical Sciences and Applications. 1992 Jun 10;577(2):305-15. References