Affinity chromatography

Affinity Chromatography

1. Affinity Chromatography Maryam Yekefallah Fall 2017

2. Content Introduction of chromatography Different aspects of affinity chromatography The principle of Affinity Chromatography Procedure Application Advantages & Disadvantages

3. Chromatography Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (immobilize phase) while the other (the mobile phase) moves in a definite direction

4. Affinity History 1930s, first developed by A.Wilhelm Tiselius-a Swedish biochemist, won the Nobel Prize in 1948 Used to study enzymes and other proteins Relies on the affinity of various biochemical compounds with specific properties

5. Introduction 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 a method of separating a mixture of proteins or nucleic acids by specific interactions of those molecules with a component known as a ligand , which is immobilized on a support. M ixture of proteins is passed 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).

6. Introduction The other proteins in the solution wash through the column because they were not able to bind to the ligand.

7. Specificity of Affinity Chromatography Specificity is based on three aspects of affinity Matrix Spacer arm Ligand

8. 1- Matrix The matrix simply provides a structure to increase the surface area to which the molecule can bind. Amino, hydroxyl and carbonyl groups located with the matrix serve as ligand binding sites. Matrix are made up of agarose and other polysaccharides

9. Matrix For having an effective matrix, it must have certain characters: 1)It must be insoluble in solvents and buffers employed in the process 2)It must be chemically and mechanically stable . 3)It must be easily coupled to a ligand or spacer arm onto which the ligand can be attached . 4)It must exhibit good flow propertie s and have a relatively large surface area for attachment

10. Types of Matrix used Cellulose : used for DNA affinity chromatography. Agarose : DNA or proteins Tris- acryl It is having higher separation ability. It has small particle size 40-80 micrometer .

11. 2- Spacer arm The stationary phase is typically a gel matrix, to prevent steric interference or overlap during the binding process of the target molecule to the ligand, an inhibitor containing a hydrocarbon chain is first attached to the agarose bead (solid support). This inhibitor with a hydrocarbon chain is commonly known as the spacer between the agarose bead and the target molecule.

12. 3- Ligand The Ligand binds only to the desired molecule within the solution It attaches to the matrix which is made up of an inert substance It should only interact with the desired molecule and form a temporary bond The ligand/molecule complex will remain in the column, eluting everything else off The ligand/molecule complex dissociates by changing the pH

13. Ligand The ligand can be selected only after the nature of the macromolecule to be isolated is known. Examples: For antibody isolation ,an antigen may be used as ligand . If an enzyme is to be purified , a substrate analog, inhibitor, cofactor, or effector may be used as a the immobilized ligand.

14. The principle of Affinity Chromatography The Sample is injected into the equilibrated affinity chromatography column Only the substance with affinity for the ligand are retained on the column The substance with no affinity to the ligand will elute off The substances retained in the column can be eluted off by changing the pH of salt or organic solvent concentration of the eluent

15. We have 3 main part:

16. Procedure Step-1 Attach ligand to column matrix Binding of the selected ligand to the matrix requires that a covalent bond be formed between them. Most ligands are attached first to spacer arms which are then bonded to the matrix. The ligand-matrix gel is then loaded into an elution column.

17. Step 2: Load protein mixture onto column Once the column has been prepared, the mixture containing isolate is poured into the elution column. Gravity pulls the solution through the gel, because most of the proteins do not bind to the ligand-matrix complex. When ligand is recognized substrate passes through the gel, it binds to the ligand-matrix complex, halting its passage through the gel. Some of the impurities flow through the gel due to gravity, but most remain, unbound, in the gel column

18. Step 3: Proteins bind to ligand In order to remove these unbound impurities, a wash of extreme pH, salt concentration, or temperature is run through the gel. It is important to use a strong wash so that all the impurities are removed. Once the impurities are washed-out, the only remaining part of the protein mixture should be the desired isolates.

19. Step 4: Wash column to remove unwanted material In ally to collect isolate, which is still bound to the ligand-matrix in the gel, a stronger second wash is run through the column.

20. Step 5: Wash off proteins that bind loosely This second wash relies on the reversible binding properties of the ligand, which allows the bound protein to dissociate from its ligand in the presence of this stronger wash.

21. Step 6: Elute Proteins that bind tightly to ligand and collect purified protein of interest The protein is then free to run through the gel and be collected.

22. Application 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

23. 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.

24. Industrial Application Affinity chromatography is widely used in the pharmaceutical industry to purify and extract molecules of interest from complex mixtures. These molecules tend to be enzymes, proteins or amino acids, but other biological species can be selectively retained.

25. Antibody affinity (Immune affinity Chromatography) Used to purify antibody against a specific antigen Ex : Immunoglobulins

26. Others Pregnancy test Allergy test Immune assay Kinetic studies Qualitative measurement of substrate.

27. 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

28. Disadvantages of affinity chromatography Expensive ligands Leakage of ligand Degradation of the solid support Limited lifetime Non-specific adsorption

29. Reference 1- Cuatrecasas , Pedro (June 25, 1970).  "Protein Purification by Affinity Chromatography" (PDF).  JBC . Retrieved November 22, 2017 . 2-   Uhlén M (2008).  "Affinity as a tool in life science" . Biotechniques.  44  (5): 649–54.  doi : 10.2144/000112803 .  PMID   18474040 . 3-   Hage , David (May 1999).  "Affinity Chromatography: A Review of Clinical Applications"  (PDF). Clinical Chemistry.  45  (5): 593–615.