Thursday, August 5, 2010

Week 6


Dr. Clendening and I had decided late last week that we would start a new project on Monday. We wanted to investigate the protein WT1. WT1 seemed like a good candidate for two reasons. First of all, we knew that WT1 worked synergistically with SF1 to promote the downstream expression of SOX9 and AMH, eventually leading to testes differentiation. Secondly, we knew that WT1 had two different splice sites, resulting in four different isoforms. These isoforms, we predicted, might be regulated according to temperature.

Dr. Clendening and I spent the beginning of the week isolating total RNA samples from both the adrenal/kidney and gonadal tissues. I was not the easiest task, because we had to make sure that we were working in a clean environment. Everything we used had to be treated with RNAse Zap, a chemical that destroys ribonucleases. Luckily, the samples that we extracted were good. An agarose gel gave us the results we were looking for.

DAX1 and SF1 (return to the agarose gel)

Now I know that I had posted a comment in an earlier blog stating that Dr. Clendening and I wouldn't be using agarose gels, but...things weren't working well with the IEF gel. After doing some research, we found a protocol for native blotting of proteins using agarose. However, this protocol called for a different type of agarose, a high resolution blend (HRB). We had a bottle of the HRB in the lab, so we decided to go ahead and try it. I prepared samples, and ran a gel during the remainder of the week. The western provided the following results.

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