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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 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.
Affinity Ligand Matrix The resin
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.
Step 3. Proteins interact with affinity ligand with some binding loosely and others tightly.
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
Purity test SDS-PAGE Mass spectrometry N-terminal sequencing, etc.
Downstream of protein purification • Biophysical characterization • Biochemical analysis of activities • Physiological relevance • Pathological mechanisms • etc.
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
IR spectroscopy Electron microscopy
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.