Affinity chromatography


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Affinity chromatography

  2. 2. CHROMATOGRAPHY <ul><li>Chromatography is the collective term for a set of lab orator y techniques for the separation of mixtures. </li></ul>
  3. 3. <ul><li>The term comes from Gree k χρώμα: chroma means color and γραφειν: graphein means write </li></ul>
  4. 4. INVENTED BY <ul><li>1903 – Tswett, a Russian botanist coined the term chromatography. </li></ul>
  5. 5. CHROMATOGRAPHY <ul><li>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 </li></ul>
  6. 6. <ul><li> An animation </li></ul><ul><li> </li></ul>
  7. 7. TERMINOLOGY <ul><li>An immobilized phase is a stationary </li></ul><ul><li>phase which is immobilized on the support </li></ul><ul><li>particles, or on the inner wall of the column </li></ul><ul><li>tubing </li></ul>
  8. 8. TERMINOLOGY <ul><li>The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC ) , a gas (GC), or a supercritical fluid (SFC). A better definition:The mobile phase consists of the sample being s eparated and the solvent that moves the sample through the column. </li></ul>
  9. 9. TERMINOLOGY <ul><li>The analyte is the substance that is to </li></ul><ul><li>be separated during chromatography. </li></ul>
  10. 10. TERMINOLOGY <ul><li>The sample is the matter analysed in chromatography. It may consist of a single component or it may be a mixture of components. </li></ul>
  11. 11. TERMINOLOGY <ul><li>E lution </li></ul><ul><li>- washing of the mixture </li></ul><ul><li>Eluent </li></ul><ul><li>- additional solvents used for elution </li></ul><ul><li>Residency </li></ul><ul><li>- time spent on column </li></ul>
  12. 12. Types of Chromatography
  14. 14. Affinity History <ul><li>1930s, first developed by A . Wilhelm Tiselius -a swedish biochemist , won the Nobel Prize in 1948 </li></ul><ul><li>Used to study enzymes and other proteins </li></ul><ul><li>Relies on the affinity of various biochemical compounds with specific properties </li></ul>
  15. 15. Examples <ul><ul><li>Antigen Antibody </li></ul></ul><ul><ul><li> Antibody Antigen </li></ul></ul><ul><ul><li>Substrate Enzyme </li></ul></ul><ul><ul><li>DNA Histon </li></ul></ul><ul><ul><li>Hormone Binding Protein/Receptor </li></ul></ul>
  16. 16. Specificity of Affinity Chromatography <ul><li>Specificity is based on three aspect of affinity </li></ul><ul><li> Matrix : for ligand attachment. </li></ul><ul><ul><ul><ul><ul><li>Spacer arm : used to bind ligand to matrix </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Ligand : molecule that binds reversibly to a specific target molecule(site of interaction) </li></ul></ul></ul></ul></ul>
  17. 17. An animation Affinity /.../GE_ Affinity .swf -
  18. 18. So now what…. <ul><li>The Sample is injected into the equilibrated affinity chromatography column </li></ul><ul><li>Only the substance with affinity for the ligand are retained on the column </li></ul><ul><li>The substance with no affinity to the ligand will elute off </li></ul><ul><li>The substances retained in the column can be eluted off by changing the pH of salt or organic solvent concentration of the eluent </li></ul>
  19. 19. Matrix <ul><li>The matrix simply provides a structure to increase the surface area to which the molecule can bind </li></ul><ul><li>The matrix must be activated for the ligand to bind to it but still able to retain it’s own activation towards the target molecule </li></ul>
  20. 20. Matrix <ul><li>Amino, hydroxyl, carbonyl and thio groups located with the matrix serve as ligand binding sites </li></ul><ul><li>Matrix are made up of agarose and other polysaccharides </li></ul><ul><li>The matrix also must be able to withstand the decontamination process of rinsing with sodium hydroxide or urea </li></ul>
  21. 21. Ligand <ul><li>The Ligand binds only to the desired molecule within the solution </li></ul><ul><li>The ligand attaches to the matrix which is made up of an inert substance </li></ul><ul><li>The ligand should only interact with the desired molecule and form a temporary bond </li></ul><ul><li>The ligand/molecule complex will remain in the column, eluting everything else off </li></ul><ul><li>The ligand/molecule complex dissociates by changing the pH </li></ul>
  22. 22. Antibody affinity (Immunoaffinity Chromatography) <ul><li>Used to purify antibody against a specific antigen </li></ul><ul><li>Ex:Immunoglobulins </li></ul><ul><li>P urification of IgG , IgG fragments and subclasses have the high affinity of protein A and protein G for the Fc region of polyclonal and monoclonal IgG-type antibodies </li></ul>
  23. 23. Protein A and protein G <ul><li>Protein A and protein G are bacterial cell surface proteins (from Staphylococcus aureus and Streptococcus respectively) </li></ul><ul><li>Recombinant protein A is available; </li></ul><ul><li>Engineered to include a C-terminal </li></ul><ul><li>Results in an enhanced binding capacity </li></ul>
  24. 24. AFFINITY CHROMATOGRAPHY <ul><li>Can be used; </li></ul><ul><li>Purify and concentrate a substance from a mixture into a buffering solution </li></ul><ul><li>Reduce the amount of a substance in a mixture </li></ul><ul><li>Discern what biological compounds bind to a particular substance, such as drugs </li></ul><ul><li>Purify and concentrate an enzyme solution </li></ul>
  25. 25. Applications <ul><li>Used in Genetic Engineering </li></ul><ul><li>- nucleic acid purification </li></ul><ul><li>Production of Vaccines </li></ul><ul><li>- antibody purification from blood serum </li></ul><ul><li>And Basic Metabolic Research </li></ul><ul><li>- protein or enzyme purification from cell free extracts </li></ul>
  26. 26. <ul><li>Avidin(or strepdavidin) -biotin interaction is used to purify proteins </li></ul><ul><li>Avidin :protein deposited in the whites of eggs(birds,reptiles…) </li></ul><ul><li>Streptavidin is a tetrameric protein purified from the bacterium Streptomyces avidinii </li></ul><ul><li>Biotin: ( vitamin H or B7) cofactor in the metabolism of fatty acids and leucine, and in gluconeogenesis </li></ul>PROTEIN PURIFICATION
  27. 27. <ul><li>The non-covalent bond formed between biotin and avidin or streptavidin </li></ul><ul><li>has a binding affinity >most antigen and antibody bonds </li></ul><ul><li>~ strength of a covalent bond </li></ul>
  28. 28. biotinylation <ul><li>means affinity chromatography using immobilized avidin or streptavidin </li></ul><ul><li>to separate the biotinylated protein from a mixture of other proteins and biochemicals </li></ul>
  29. 29. Nucleic acid separation using immobilized metal affinity chromatography (IMAC) <ul><li>The method can be used to purify compounds </li></ul><ul><li>containing purine or pyrimidine moieties where </li></ul><ul><li>the purine and pyrimidine moieties are shielded </li></ul><ul><li>from interaction with the column matrix from </li></ul><ul><li>compounds containing a non-shielded purine or </li></ul><ul><li>pyrimidine moiety or group. </li></ul>
  30. 30. <ul><li>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 </li></ul><ul><li>IMAC columns clarify plasmid DNA from bacterial alkaline lysates, purify a ribozyme, and remove primers and other contaminants from PCR reactions </li></ul>
  31. 31. ADVANTAGES OF AFFINITY CHROMATOGRAPHY <ul><li>Extremely high specificity </li></ul><ul><li>High degrees of purity can be obtained </li></ul><ul><li>The process is very reproducible </li></ul><ul><li>The binding sites of biological molecules can be simply investigated </li></ul>
  32. 32. DISADVANTAGES OF AFFINITY CHROMATOGRAPHY <ul><li>1) Expensive ligands </li></ul><ul><li>2) Leakage of ligand </li></ul><ul><li>3) Degradation of the solid support </li></ul><ul><li>4) Limited lifetime </li></ul><ul><li>5) Non-specific adsorption </li></ul><ul><li>6) Relatively low productivity </li></ul>
  33. 33. REFERENCES <ul><li>[1] </li></ul><ul><li>[2] Affinity %20 Chromatography %201.ppt </li></ul><ul><li>[3] - </li></ul><ul><li>[ 4] Affinity %20 Chromatography .pdf - </li></ul>
  34. 34. <ul><li>THANKS… </li></ul>