Affinity chromatography

B pharm 7th sem industrial method of analysis

1. Assignment on : Affinity Chromatography Submitted to: Prof. (Dr.) P . Malairajan Head Of Department Submitted By: Name: Netra Prasad Neupane Bachelor of pharmacy (7 th sem ) Id No. 17BPH080 Subject: instrumental method of analysis (701P) Sam Higginbottom University of Agriculture, Technology and Sciences, P rayagraj, UP 1

2. Acknowledgment In preparation of my assignment, I had to take the help and guidance of some respected seniors and professors, who deserve my deepest gratitude. As the completion of this assignment gave me much pleasure. I would like to show my gratitude to Prof . (Dr.) P. Malairajan (HOD, SHIATS.FHS,SHUATS) Course Instructor. He gave me wonderful opportunity to prepare assignment on “AFFINITY CHROMATOGRAPHY” topics. I would also like to expand my gratitude to all those who have directly and indirectly guided me in writing this assignment. Many people, especially my parents and classmates have made valuable comment suggestions on my assignment which gave me an inspiration to improve the quality of the assignment 2

3. Tablet of contents S.N CONTENTS 1. Introduction and definition to affinity chromatography 2. Common term use in affinity chromatography, Principle of affinity chromatography 3. Components of affinity chromatography 4. Step involves in affinity chromatography 5. Application, advantage and disadvantage 6. Conclusion and References 3

4. Introduction to affinity chromatography Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatography matrix. The technique offers high selectivity, hence high resolution, and usually high capacity for the protein(s) of interest. Purification can be in the order of several thousand-fold and recoveries of active material are generally very high. 4

5. Definition of affinity chromatography Affinity chromatography is unique in purification technology since it is the only technique that enables the purification of a biomolecule on the basis of its biological function or individual chemical structure. The technique can be used to separate active bimolecule from denatured or functionally different forms, to isolate pure substances present at low concentration in large volumes of crude sample and also to remove specific contaminants. 5

6. Common terms in affinity chromatography Matrix: for ligand attachment. Matrix should be chemically and physically inert. Spacer arm: used to improve binding between ligand and target molecule by overcoming any effects of steric hindrance. Ligand: molecule that binds reversibly to a specific target molecule or group of target molecules. Binding: buffer conditions are optimized to ensure that the target molecules interact effectively with the ligand and are retained by the affinity medium as all other molecules wash through the column. 6

7. Elution: buffer conditions are changed to reverse (weaken) the interaction between the target molecules and the ligand so that the target molecules can be eluted from the column. Wash: buffer conditions that wash unbound substances from the column without eluting the target molecules or that re-equilibrate the column back to the starting conditions (in most cases the binding buffer is used as a wash buffer). Ligand coupling: covalent attachment of a ligand to a suitable pre-activated matrix to create an affinity medium. Pre-activated matrices: matrices which have been chemically modified to facilitate the coupling of specific types of ligand. 7

8. Principle of Affinity Chromatography The stationary phase consists of a support medium, on which the substrate (ligand) is bound covalently, in such a way that the reactive groups that are essential for binding of the target molecule are exposed. As the crude mixture of the substances is passed through the chromatography column, substances with binding site for the immobilized substrate bind to the stationary phase, while all other substances is eluted in the void volume of the column. Once the other substances are eluted, the bound target molecules can be eluted by methods such as including a competing ligand in the mobile phase or changing the pH, ionic strength or polarity conditions. 8

9. Components of Affinity Chromatography 1) Matrix The matrix is an inert support to which a ligand can be directly or indirectly coupled. In order to for the matrix to be effective it must have certain characters: Matrix should be chemically and physically inert. It must be insoluble in solvents and buffers employed in the process It must be chemically and mechanically stable. 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. The most useful matrix materials are agarose and polyacrylamide. 9

10. 2) Spacer arm It is used to improve binding between ligand and target molecule by overcoming any effects of steric hindrance. 3) Ligand It refers to the molecule that binds reversibly to a specific target molecule. 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 ligand. If an enzyme is to be purified, a substrate analog, inhibitor, cofactor, or effectors may be used as a the immobilized ligand. 10

11. Steps in   Affinity Chromatography Affinity medium is equilibrated in binding buffer. Sample is applied under conditions that favor specific binding of the target molecule(s) to a complementary binding substance (the ligand). Target substances bind specifically, but reversibly, to the ligand and unbound material washes through the column. 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. Affinity medium is re-equilibrated with binding buffer. 11

12. These events can be summarized into the following three major steps: 1) Preparation of Column The column is loaded with solid support such as sepharose , agarose, cellulose etc. Ligand is selected according to the desired isolate. Spacer arm is attached between the ligand and solid support. 2) Loading of Sample Solution containing a mixture of substances is poured into the elution column and allowed to run at a controlled rate. 3) Elution of Ligand-Molecule Complex Target substance is recovered by changing conditions to favor elution of the bound molecules. 12

13. Applications and uses of affinity chromatography. 1) Immunoglobulin purification (antibody immobilization) 2) Recombinant tagged proteins. 3) Protein A, G, and L purification 4) Biotin and biotinylated molecules purification 5) Affinity purification of albumin and macroglobulin contamination. 6) Separation of mixture of compounds. 7) Detection of Single Nucleotide polymorphisms and mutations in nucleic acids 8) In enzyme assays 9) Detection of substrates 10) Investigation of binding sites of enzymes 13

14. Advantages of Affinity Chromatography High specificity Target molecules can be obtained in a highly pure state Single step purification The matrix can be reused rapidly. The matrix is a solid, can be easily washed and dried. Give purified product with high yield. Affinity chromatography can also be used to remove specific contaminants, such as proteases. Disadvantage Time consuming method. More amounts of solvents are required which may be expensive. Intense labour 14

15. Conclusion : In above, details study about affinity chromatography, Common term use in affinity chromatography, Principle of affinity chromatography , Components of affinity chromatography , Step involves in affinity chromatography, Application, advantage and disadvantage has been done. References : 1) https://microbenotes.com/affinity-chromatography/ (accessed date 02/09/2020) 2) http://cdn.intechopen.com/pdfs/33046/InTech Affinity_chromatography_principles_and_applications.pdf (accessed date 02/09/2020) 15