Difference between revisions of "Affinity Purification of Proteins - FPLC"

Revision as of 13:47, 13 September 2010

Preparation of Lysates and Buffers

• Lysates can be prepared by any means, but must be filtered through a 0.22 micron filter prior to applying to the column.
• Filter all buffers through a 0.22 micron filter. You need an extra ~50 mL of buffer for priming the pumps

HPLC Setup

1. Check that the HPLC is set up for protein work and not radioactive work (see HPLC - Plumbing Setup)
2. Turn on computer, pumps and lamp if not already on by pressing the buttons on the front.
3. Using TC Navigator, attach the HPLC (if not attached)
4. Disconnect the column from the top (if already connected)
5. Load the desired method, or write a new one (see HPLC - Designing a Method for a Sample or Series)
6. Go to setup and turn the pump on.
7. Rinse the buffer leads with water and place into the appropriate buffers (see the setup page for which lead corresponds to which buffer).
8. Prime the pumps in the following order by unscrewing the black wheel in the pump (remove the panel) and sucking up ~25 mL of liquid with a 60 mL syringe:
   1. Protein sample (if the protein is being applied via the pumps)
   2. Elution buffer
   3. Wash buffer(s)
9. Let the equillibration/wash buffer run for a few minutes
10. Connect the inlet tube to the top of the affinity column (it should still be running wash/equillibration buffer) using the appropriate fittings
11. Connect the bottom of the column to the tube leading into the UV monitor
12. Check all connections for leaks.
13. Set up the Fraction Collector as desired
14. Check the absorbance (normally 280nm) on the setup panel to ensure that it is ~0. If it is not, let it equillibrate a few more minutes and check it again. If the absorbance is >0.3 then the buffer is absorbing too much light at 280nm. If that is the case, use a method with no UV and turn off the UV lamp from the setup panel.
15. Once everything is ok (and the status panel is marked ready), start the run by turning the injector port to load. If you are loading sample via the pump, either port will work (and can be turned immediately back to the load position). If you are loading via the sample loops, wait until 5 times the loop volume has passed through before turning back.
16. Check your progress by clicking the real time monitor button.