1. Purification of GFP using HIC Chromatography

2. Chromatography • A technique used to separate molecules based on how they tend to cling to or dissolve in various solids, liquids, and gases.

3. Characteristics of Proteins • Hydrophilic • Hydrophobic • Positive • Negative • Size • Active site

4. Types of Chromatography • Size –Exclusion: separation based on size • Ion-Exchange: separation based on charge • Affinity: Lock and key interaction between protein and column matrix • Hydrophobic Interaction: separate proteins using various salt concentrations

5. Size Exclusion

6. Gel Filtration Chromatography

7. Ion Exchange

8. Affinity

9. Hydrophobic Interaction

10. GFP Purification • GFP bacteria cells are broken open using a detergent. • The cell contents are washed with a high-salt binding buffer. The charged ions in the salt repel the ions on the exterior of the GFP protein which results in the protein turning itself inside out. • The exposed GFP binds to the resin beads in the column.