Presentation on theme: "PURIFICATION OF GFP USING HIC CHROMATOGRAPHY. Chromatography A technique used to separate molecules based on how they tend to cling to or dissolve in."— Presentation transcript:
Chromatography A technique used to separate molecules based on how they tend to cling to or dissolve in various solids, liquids, and gases.
Characteristics of Proteins Hydrophilic Hydrophobic Positive Negative Size Active site
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
GFP Purification 1. GFP bacteria cells are broken open using a detergent. 2. 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. 3. The exposed GFP binds to the resin beads in the column.