1. Developing A Protein Purification Protocol Billie Parker 6-14-02

2. Overview  Introduction to Chromatography  Extraction of Protein from Cells  Introduction to Green Fluorescent Protein  Results  Conclusions

3. Introduction to Chromatography  Chromatography is used to purify complex mixtures.  We used a resin in a column.  The resin binds to certain proteins. This purifies these proteins away from other proteins that do not bind to the resin.  After the other proteins have passed through, the bound proteins can be released.  There are different resins for different substances you want to purify.

4. Hydrophobic Interaction  The resin binds to water-hating proteins.  Increased salt concentrations promote binding.  The bound protein can be released by lowering the salt.  We tried three different resins to see which one worked the best.

5. Column Information  The resin is packed into the column already.  The substance to be purified passes through the resin and binds to it.  Most molecules do not bind to the resin.

6. FPLC System Components  The buffers carry the sample through the system.  There are two pumps to move the buffers.  Sample is injected into the valve.  Then the sample passes to the column.  The UV light detector measures the amount of protein based on absorbance.  The sample is divided into fractions by the fraction collector.

7. FPLC System

8. Extraction of Protein from Cells  Getting protein out of cells is the first step in purification.  We centrifuged the bacteria to get them out of the growth medium.  We used two different methods to break open the bacteria.  We resuspended the cells in detergent.  We resuspended the cells and froze them.

9. Centrifuge

10. Extraction by Detergent  After lysing the cells in detergent, we centrifuged the cells at 10,000 xg for 10 minutes and kept the supernatant.  We used a particular resin that binds to only detergent to purify the detergent from the supernatant.  We put ammonium phosphate in the extract to increase the amount of salt to promote binding to the resin.  Then, we loaded the extract on the column.

11. Extraction by Freezing  We used TE Buffer to resuspend the cells.  We put the cultures into the freezer at -70 o for 20 minutes.  We let the cells thaw.  Then we centrifuged at 10,000 xg for 10 minutes and kept the supernatant.  We put ammonium phosphate in the extract to increase the amount of salt to promote binding to the resin.  Then, we loaded the extract on the column.

12. Introduction to GFP  GFP is Green Fluorescent Protein.  GFP is from jellyfish.  We used bacteria that were engineered to make GFP.  GFP glows when you shine UV light on it.

13. Introduction to GFP  GFP is Green Fluorescent Protein.  GFP is from jellyfish.  We used bacteria engineered to make GFP.  GFP glows when you shine UV light on it.

14. Results  The graphs will show the amount of protein indicated by UV absorbance.  The salt concentration starts high to allow the GFP to bind then it decreases to let the GFP come out.  GFP was detected by shining UV light on the collected fractions.

15. First Column Tested  Salt shows the salt concentration in the buffer.  UV measures total protein.  GFP shows fractions that glow.

16. Second Column Tested  Salt shows the salt concentration in the buffer.  UV measures total protein.  GFP shows fractions that glow.

17. Third Column Tested  Salt shows the salt concentration in the buffer.  UV measures total protein.  GFP shows fractions that glow.

18. Conclusions  The octyl column did not bind to the GFP.  Both the phenyl and butyl columns bound the GFP and eluted it with low salt.  We were unable to conclude which column worked the best because we cannot tell how much protein is in the GFP fractions.