AFFINITY chromatography.pptx

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1. AFFINITY CHROMATOGRAPHY

2. What is Affinity Chromatography? Chromatographic technique for selective separation/purification of a molecule from a biochemical mixture. PRINCIPLE Separation of mixture of protein/nucleic acids/enzymes take place by specific and reversible interaction of these molecules with a component – Ligand , which is immobilized on an inert support, and packed in a column When a mixture of, say, proteins passes through the column, one of the proteins binds to the ligand based on its specificity and high affinity The protein and the ligand fit together like a lock and key Other proteins in the mixture pass through the column since these are unable to bind to the ligand

3. Components of Affinity Matrix Matrix Spacer Target protein Affinity Matrix Highly selective Ligand

4. Matrix or Affinity Supports Inert support to which ligand is directly or indirectly bound Chemically and mechanically stable Uniform particle and pore size Low non specific adsorption Examples: Cellulose, agarose , silica, polystyene , sepharose (All these are available commercially)

5. Ligand Selection based on specific and reversible binding with the target molecule Carries a group which can couple to matrix without losing binding activity Stable in different binding and elution conditions Ligand can be bound to matrix by Covalent bonding Non-covalent bonding Adsorption Biospecific interaction

6. Commonly used Ligands Ligand Specificity Blue B Kinases , dehydrogenases , nucleic acid binding proteins Orange A Lactate dehydrogenase Lysine Plasminogen , rRNA , dsDNA Protein A Fc regions of many IgG subtypes; species dependent weak interactions with IgA , IgM , IgD Biotin Streptavidin , avidin Gelatin Fibronectin Lectins Glycoproteins , polysaccharides, glycolipids Direct binding of ligand to matrix may lead to stearic hindrance resulting in inefficient binding of target to ligand . Solution: Spacer

7. Spacer Overlap between target molecule and bead leads to inefficient binding ,i.e., stearic hindrance Spacer covalently bound to matrix Allows complete access of target molecule to ligand . Should be hydrophillic Should be of optimum length Matrix

8. Steps Involved in Affinity Chromatography Matrix beads + Buffer: Beads swell Couple the ligand (and spacer, if required) with the matrix Filter: Affinity matrix Pack in a glass column Equilibrate the column with buffer Load the sample Wash the column to remove unbound molecules Elute bound molecules Analyze the eluent

9. 1.Binding 2. Washing 3. Elution Ligand Other proteins Target protein Steps Involved in Affinity Chromatography

10. Elution Specific Elution - Competing free ligand - Competing binding substance Competitive ligand in solution + Example: Elution of enzymes from Blue Sepharose by free NADH Competitive binding substance + Example: Elution of Antigens from antibody columns with specific peptides Competitive soluble ligands or binding substances can elute the bound target specifically Gentler than general methods Expensive (like specific peptides for antigen elution)

11. Applications Purification of substances from biological mixtures. Separation of native form of protein from denatured form. Purification and concentration of enzymes in solution.

12. Gel Chromatography

13. Liquid chromatography PRINCIPLE: Separates molecules in solution by their “effective size” in solution. Hence, also called size exclusion chromatography. Separation is achieved by the differential exclusion or inclusion of solutes as they pass through porous stationary phase consisting of cross linked polymeric gel beads. Gel Chromatography Also known as gel filtration/gel permeation/size exclusion chromatogaphy (GFC) (GPC) (SEC)

14. Gel Chromatography Stationary Phase Semi permeable, porous polymer gel beads. Properties of gel beads Chemically inert Mechanically stable Uniform particle and pore size Examples Dextran gel Agarose gel Polyacrylamide gel

15. Advantages Short analysis time No sample loss Less consumption of mobile phase Disadvantage Molecules with closely related molecular mass show broad peak

16. Ultra High Performance Liquid Chromatography

17. UHPLC v/s HPLC Characteristics UHPLC HPLC Particle size <2 µm 5-7 µm Pressure 20000 psi 500-6000 psi