1. Protein Structure and Analysis
Activity 7.3: GFP Purification
by Hydrophobic Interaction
Chromatography

2. Activity 7.3: GFP Purification by Hydrophobic Interaction Chromatography
Research question:
Can GFP be separated from a mixture of proteins using HIC?
Objectives:
Use aseptic technique to inoculate LB/amp/ara broth with bacteria transformed with the pGLO plasmid
Grow liquid cultures overnight
Centrifuge the bacterial culture and lyse bacteria
Centrifuge the bacterial cell lysate to isolate the supernatant
Apply the supernatant to the HIC column
Apply buffers with different salt concentrations to the column
Collect fractions

3. Safety Reminders Follow all laboratory safety procedures
Read and become familiar with MSDS for the activity
Inform the instructor if you are allergic to antibiotics
Use aseptic technique when handling bacteria and dispose of microbial waste properly
Wear appropriate PPE

4. Skills to Master
Aseptically inoculate a liquid culture from a plate (Activity 3.3)
Make a bacterial lysate from a liquid culture
Perform HIC
Use a microcentrifuge (Activity 5.3)

5. Tips
When removing the end of the column for the first time, do not twist, snap sharply at a 90º angle
This will create a large opening in the column
Insert a small wedge of paper between the column and the collection tube to prevent an airtight seal between column and tube
Insert transfer pipet as far as possible into the column without touching the resin when delivering the sample. Add slowly as to not disturb the resin

6. Tips
Make sure that the bacteria transformed with pGLO (from Activity 5.2) are viable. Approximately 24 to 72 hours ahead of the laboratory, re-streak the transformed bacteria from stab cultures, glycerol stocks, or agar plates (less than 2 weeks old) onto fresh LB/amp/ara plates
Once the liquid cultures are centrifuged, resuspend the pellet thoroughly in TE to ensure that the lysozyme and freeze-thaw cycling will lyse the cells
Track the GFP during the process by using a UV light

7. Student Workstation Materials

8. Student Workstation Materials

9. Purification of GFP
Play video: HIC Chromatography

10. Protocol Highlights/Tips
Follow aseptic technique
Resuspend pellet to ensure lysis
Make sure to snap end off column at 90º to ensure large opening
Add samples slowly near resin
Add samples slowly to prevent disruption of the resin bed
Collect fractions in collection tubes, use a wedge of paper to prevent airtight seal

11. Results
Collect the fractions into the tubes

12. Summary Make sure to: Record all steps in your notebook
Follow aseptic technique at all times when working with bacteria
Resuspend well to ensure proper lysate production
Use a wedge of paper to prevent airtight seal of column and collection tubes
Wear UV protection when observing the GFP with a UV light