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. Matrix Affinity Ligand The resin

4. Examples of tags and ligands His-tag FLAG TM peptide Strep-tag GST tag Maltose binding protein fusion Calmodulin binding protein fusion Transition metal ion Monoclonal antibody Biotin Glutathione Amylose Ca 2+ 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. Native Unfolded Fluorescence spectroscopy Trp68 Trp42 Trp156 Trp130 N-terminal domainC-terminal domain Diagrams of crystal structure of HgD Example: Human lens crystallins Human gene cloned into pET vector and expressed in E. coli.

15. Protein folding mechanisms

16. Electron microscopy IR spectroscopy

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 Three groups of properties of the target molecule are used in affinity chromatography: 1. Specific binding properties based on biological activity like: - Enzyme active sites - Receptor binding sites - Antibody binding sites etc. These are used together with the natural ligand or an analogue of it. Sometimes the analogue has a broader specificity and can be used for group separations. 2. Naturally occurring prosthetic groups like: polysaccharides etc. Such properties normally allow group separations only. 3. Molecules equipped with an affinity tag like: - Glutathione-S-Transferase (GST) - Oligo histidine etc. This group of properties is used almost exclusively for recombinant fusion proteins.