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Affinity Chromatography

Affinity Chromatography. Yongting Wang Jan07. What is AC?. Affinity chromatography (AC) is a technique enabling purification of a biomolecule with respect to biological function or individual chemical structure. AC is designed to purify a particular molecule from a mixed sample.

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Affinity Chromatography

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  1. Affinity Chromatography Yongting Wang Jan07

  2. What is AC? • Affinity chromatography (AC) is a technique enabling purification of a biomolecule with respect to biological function or individual chemical structure. • AC is designed to purify a particular molecule from a mixed sample.

  3. Affinity Ligand Matrix The resin

  4. His-tag FLAGTM peptide Strep-tag GST tag Maltose binding protein fusion Calmodulin binding protein fusion Transition metal ion Monoclonal antibody Biotin Glutathione Amylose Ca2+ Examples of tags and ligands There are situations where you don’t need a tag.

  5. Step 1. Loading affinity column.

  6. Step 2. Proteins sieve through matrix of affinity beads.

  7. Step 3. Proteins interact with affinity ligand with some binding loosely and others tightly.

  8. Step 4. Wash off proteins that do not bind.

  9. Step 5. Wash off proteins that bind loosely.

  10. Step 6. Elute proteins that bind tightly to ligand and collect purified protein of interest. http://www.bio.davidson.edu/Courses/genomics/method/Affinity.html

  11. Affinity chromatography applied to recombinant proteins

  12. Purity test SDS-PAGE Mass spectrometry N-terminal sequencing, etc.

  13. Downstream of protein purification • Biophysical characterization • Biochemical analysis of activities • Physiological relevance • Pathological mechanisms • etc.

  14. Trp68 Trp156 Trp42 Trp130 N-terminal domain C-terminal domain Example: Human lens crystallins Human gene cloned into pET vector and expressed in E. coli. Fluorescence spectroscopy Diagrams of crystal structure of HgD Unfolded Native

  15. Protein folding mechanisms

  16. IR spectroscopy Electron microscopy

  17. In summary, Affinity chromatography is a method that will allow you to purify a particular molecule from a mixed sample so that further investigation of this molecule could be carried out. • Thank you for your attention.

  18. Types of target molecular properties

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