Week 4

Test Run

The beginning of the week was devoted to running an actual version of my experiment. I incubated samples of DAX1 and SF1 at male producing temperature (MPT), 26 degrees celsius, and female producing temperature (FPT), 31 degrees celsius. Half of the samples received a formaldehyde/glycine treatment to theoretically "fix" the proteins and all of the samples were loaded into a 4-20% polyacrylamide gel. The gel ran for an hour at 100 volts. Afterwards, the proteins were transferred to nitrocellulose membranes through electroblotting (I electroblotted twice to get two, presumably, identical membranes).

The next day I developed western blots, but the results were not satisfactory. Half of the samples did not show up (left side of photos). The lanes that did appear reacted with both DAX1 and SF1 antibodies, but these lanes should have reacted with only one or the other. It seemed as though there might have been some non-specific binding of the antibodies, so Dr. Clendening suggested that I lower the concentration of antibody in the future. We had no explanation for the lanes that did not show up. Perhaps the proteins got degraded?

I spent the middle of the week researching the protein Wilm's Tumor Suppressor (WT1). I had run out of gels, so I had to find something to do in the meantime. It turns out that WT1 plays a role in TSD. It has many different isoforms, which may or may not be regulated by temperature. WT1 would serve as a prime candidate for future experimental applications.

IEF Test Run

By the end of the week the IEF gels had arrived and I was able to return to the DAX1/SF1 experiment. I ran an IEF gel on Friday with samples of nuclear fraction, nuclear fraction with formaldehyde/glycine treatment, and DAX1 isolated. I tried three different methods for transferring: electroblot with acetic acid buffer, electroblot with tris-glycine buffer, and a pressure blot with tris-HCl buffer. The membranes were left to dry over the weekend.