Activity 7.3

1. Activity 7.3 Hydrophobic Interaction Chromatography

2. GFP Purification Procedures Overview Day 1 Day 2 Day 3

3. Why use chromatography? • To purify a single protein from thousands in E. coli? • We will purify the GFP protein. • We will isolate it from other proteins using chromatography • Other proteins will elute from column while GFP binds. • In the last step, GFP will be eluted from the column

4. Hydrophobic Interaction Chromatography Step 1 - Add bacterial lysate to column matrix in high salt buffer Step 2 - Wash less hydrophobic proteins from column in low salt buffer Step 3 - Elute GFP from column with no salt buffer

5. Hydrophobic bead H O - - H + O O S N H H O + O - - O S O - O + Add bacterial lysate to column in high salt buffer • Hydrophobic proteins interact with column • Salt ions interact with the less hydrophobic proteins and H2O

6. Hydrophobic bead + + + - - - + + + Wash less hydrophobic proteins from column in low salt buffer • Less hydrophobic E. coli proteins fall from column • GFP remains bound to the column

7. + + + + + - - - - - + + + + + Elute GFP from column using a low salt buffer Hydrophobic bead GFP • Released from column matrix • Flows through the column