Affinity chromatography

AFFINITY CHROMATOGRAPHY

1. PRESENTATION ON ROPRINCIPLE AND APPLICATIONS OF AFFINITY CHMATOGRAPHY Presented by: Guided by: Mohd Fahad Mr. Pramdeep Bagga M. Pharma Ist Year Asstt . Professor (Pharmacology) Faculty of Pharmacy INTEGRAL UNIVERSITY LUCKNOW FACULTY OF P[HARMACY

2. INTRODUCTION 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 (molecules) by specific interactions of those molecules with a component known as a ligand, which is immobilized on a support. If a solution of, say, a mixture of proteins is passed over (through) the column, one of the proteins binds to the ligand on the basis of specificity and high affinity (they fit together like a lock and key ).

3. Principle : The stationary phase is typically a gel matrix, often of agarose; a linear sugar molecule derived from algae. Usually the starting point is an undefined heterogeneous group of molecules in solution, such as a cell lysis , growth medium or blood serum . The molecule of interest will have a well known and defined property, and can be exploited during the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule becoming trapped on a solid or stationary phase or medium.

4. The stationary phase can then be removed from the mixture, washed and the target molecule released from the entrapment in a process known as elution.The other molecules in the mobile phase will not become trapped as they do not possess this property. Possibly the most common use of affinity chromatography is for the purification of recombinant proteins

5. Column step

6. Batch steps

7. A third method, expanded bed adsorption, which combines the advantages of the two methods mentioned above, has also been developed. The solid phase particles are placed in a column where liquid phase is pumped in from 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, , charge and ionic strength directly or through a gradient to resolve the particles of interest.

8. Various affinity media   Many different affinity media exist for a variety of possible uses Amino Acid- Used with a variety of serum proteins, proteins, peptides, and enzymes, as well as rRNA and dsDNA Avidin Biotin – Used in the purification process of biotin/ avidin and their derivatives. Carbohydrate Bonding – Most often used with glycoproteins or any other carbohydrate-containing substance. Carbohydrate – Used with lectins , glycoproteins , or any other carbohydrate metabolite protein.

9. Dye Ligand – This media is nonspecific, but mimics biological substrates and proteins. Glutathione – Useful for separation of GST tagged recombinant proteins. Heparin – This media is a generalized affinity ligand , and it is most useful for separation of plasma coagulation proteins, along with nucleic acid enzymes and lipases. Hydrophobic Interaction – Most commonly used to target free carboxyl groups and proteins. Immunoaffinity – Detailed below, this method utilizes antigens' and antibodies' high specificity to separate.

10. Immobilized Metal Affinity Chromatography – Detailed further below, this method uses interations between metal ions and proteins (usually specially tagged) to separate. Nucleotide/Coenzyme – Works to separate dehydrogenases , kinases , and transaminases . Nucleic Acid – Functions to trap mRNA, DNA, rRNA , and other nucleic acids/ oligonucleotides . Protein A/G – This method is used to purify immunoglobulins . Specialty – Designed for a specific class or type of protein/coenzyme, this type of media will only work to separate a specific protein or coenzyme.

11. Thus, double-stranded plasmid and genomic DNA, which has no low binding affinity can be easily separated from RNA or oligonucleotides which bind strongly to metal-charged chelating matrices IMAC columns clarify plasmid DNA from bacterial alkaline lysates, purify a ribozyme, and remove primers and other contaminants from PCR reactions

12. Applications Used in Genetic Engineering - nucleic acid purification Production of Vaccines - antibody purification from blood serum And Basic Metabolic Research - protein or enzyme purification from cell free extracts

13. biotinylation means affinity chromatography using immobilized avidin or streptavidin to separate the biotinylated protein from a mixture of other proteins and biochemicals

14. Avidin(or strepdavidin) -biotin interaction is used to purify proteins Avidin :protein deposited in the whites of eggs(birds,reptiles…) Streptavidin is a tetrameric protein purified from the bacterium Streptomyces avidinii Biotin: ( vitamin H or B7) cofactor in the metabolism of fatty acids and leucine, and in gluconeogenesis PROTEIN PURIFICATION

15. The non-covalent bond formed between biotin and avidin or streptavidin has a binding affinity >most antigen and antibody bonds ~ strength of a covalent bond

16. Nucleic acid separation using immobilized metal affinity chromatography (IMAC) The method can be used to purify compounds containing purine or pyrimidine moieties where the purine and pyrimidine moieties are shielded from interaction with the column matrix from compounds containing a non-shielded purine or pyrimidine moiety or group.

17. ADVANTAGES OF AFFINITY CHROMATOGRAPHY Extremely high specificity High degrees of purity can be obtained The process is very reproducible The binding sites of biological molecules can be simply investigated

18. DISADVANTAGES OF AFFINITY CHROMATOGRAPHY 1) Expensive ligands 2) Leakage of ligand 3) Degradation of the solid support 4) Limited lifetime 5) Non-specific adsorption 6) Relatively low productivity

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