Weeks 8 & 9:
University of Iowa Department of Biochemistry

July 15, 2013

My eighth week in Maria Spies’ lab was spent restarting a cell line of HEK293T in order to transfect it to create FANCJ; hopefully with a higher concentration this time.  The cell line was successfully restarted on Tuesday by switching to a different set of HEK293T stocks and was scaled up over the weekend in order to have enough cells to create FANCJ.  Once the cell line had been scaled up to 20 plates the transfection was carried out, cells collected, and then the protein was purified out using the same crude purification process as the last two times.  The purification was then prepped for a heparin column chromatography.  This is similar to the gel column chromatography that was done with prep 2.  The only difference is that the column is filled with Heparin instead of gel.  I am used to dealing with Heparin in a medical role where it is used as an anticoagulation treatment for strokes, Atrial Fibrillation, and other conditions.  Heparin is useful in this instance because it is a polarized molecule and thus mimics DNA’s binding characteristics which mean that something that binds to DNA is also likely to bind to Heparin.  The protein is then eluted by gradually increasing the salt concentration within the column which forces the protein to unbind.  The protein and the flag peptide unbind at different salt concentrations which allows us to separate the protein from the flag peptide.  The Heparin column will be run next week.

These two weeks have been a little stressful because of the need to restart the cell line and the knowledge that Maria’s next grant application is dependent upon having some results to show from these experiments on FANCJ.  Knowing how important this set of experiments is and that I only have a month and a half in the lab makes this feel a lot like the school year on the block plan.  It feels like home.