Week 3:
University of Iowa Department of Biochemistry

June 4, 2013

The frozen cells from the previous week were thawed and put through a process called crude filtration.  The cells were lysed and the lysate was then filtered before being mixed with beads coated in an anti-FLAG protein that will bind to the flag tag on our FANCJ and hold it to the beads.  The bead and lysate mixture was then incubated for 24 hours on an automated rotator.  The FANCJ was then washed off of the beads and the elution was run on a SDS-PAGE gel along with samples of what flowed through the beads (what didn’t bind) and a positive FANCJ control.  The gel was then processed using the Western Blot protocol and imaged.  The Western Blot showed that the eluate did contain FANCJ and that the Flowthrough didn’t contain a noticeable amount.  The next step was to run a bulk fluorometric assay on the eluate to determine its concentration next week.

Each sample was run twice on this gel.  The right side of the gel had overflow issues and so was disregarded as all samples on that side were contaminated.  The left side shows the positive control at around 150 kilodaltons and the eluate at a similar size.

The Western Blot was very similar to the Southern blot that I had done in Cell and Molecular Biology.  The tags that were used were different but the overall process was very similar.