PURIFICATION OF GFP USING HIC CHROMATOGRAPHY

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

Size Exclusion

Gel Filtration Chromatography

Ion Exchange

Affinity

Hydrophobic Interaction

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.