Week 3

Results from the Wicking Technique

The beginning of the week was focused on developing western blots from last week's wicking transfer. I followed the standard procedure for developing westerns, but only the IEF markers appeared. Furthermore, the markers were found on only one of the membranes, instead of both as expected. Dr. Clendening and I thought that the proteins may have been retained in the agarose gel, so we preformed a coomassie stain. Sure enough, there were proteins in the gel. We decided not to use agarose gels in the future.
In the middle of the week I tested the effects of the formaldehyde application. Last week's results indicated that the formaldehyde might have reacted with the extraction buffer from the NucBuster kit. To test this hypothesis, I isolated a nuclear fraction using a different protein extraction kit (presumably with different extraction buffers). This fresh sample was to be compared with a nuclear fraction from the previous week (NucBuster extraction kit). I aliquoted samples of the two nuclear fractions and gave them either the standard formaldehyde and glycine treatment or nothing at all. All of the samples were incubated on ice for 15 minutes. Upon examination, it was clear that the nuclear fraction from the NucBuster kit reacted with the formaldehyde/glycine application and formed a precipitate. The fresh sample, that was isolated from the other kit, did not appear to react. Dr. Clendening and I decided to continue with our test the next day and load samples from both extraction kits on a protein gel.

Yet, when I came into lab, there was a slight change of plans. Dr. Clendening wanted me to isolate nuclear fractions from fresh tissue using the NucBuster extraction kit. She also wanted me to isolate nuclear fractions from not only brain tissue, but also the adrenal/kidney tissue; the gel would hopefully tell us if one tissue was preferable over the other. I preformed the isolation steps and added the formaldehyde solution to half of the samples. But, when I was about to add the glycine, I noticed that it contained mold. Dr. Clendening thought that the mold might have caused the precipitate we had seen earlier in the week, so we made a new glycine solution and added it to the formaldehyde samples; it was unclear whether or not these samples formed any precipitate. We proceeded to load the samples into a 20-40% polyacrylamide gel and I successfully transferred the proteins to a nitrocellulose membrane later that day.

On Friday, I developed the western blot. Every sample showed up! Thankfully, we were able to learn many things from this gel. For one, the adrenal/kidney tissue contained more SF1 than the brain tissue. Secondly, the samples with formaldehyde/glycine were less visible than the untreated samples. They did show up though, so presumably the cross-linking experiment was feasible. Third of all, the isolated SF1 and DAX1 samples appeared, indicating that we had useful positive controls. Moreover, these samples could be used in later application to more accurately depict the interactions between SF1 and DAX1. Though, they would need to be concentrated, because they produced a faint band. Last of all, and perhaps most importantly, we recognized several bands of SF1 in the nuclear fraction. These bands indicated that SF1 was bound to some additional protein(s) or other nuclear component(s). Since our research was based upon the hypothetical binding of SF1 and DAX1, two different proteins, this evidence was rather encouraging.