the future

There is a lot to do once the semester starts.  First, I need to quantify the results from my ChIP so I can say exactly how big of an effect Slx5 has.  After that I want to several more ChIPs on different things. For example, would I see the same effect if I removed the SUMOylation sites from htt. Also, I could do a ChIP on various truncation mutants of Slx5 to see which domains of the protein are necessary for it to remove htt from DNA. Together I think these experiments would tell me a lot about the relationship between Slx5 and htt and help me build a complete model.

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It worked!

I finally got my ChIP to work! Both my negative and positive controls worked. My results show that Slx5 reduces the amount of htt bound to DNA at the reporter gene’s promotor but not at a random centrosome spot. This is good because it confirms my model and means I am on the right research track. Interestingly, I saw the effect for all three antibodies I used but to varying degrees which may imply that some sections of the htt proteins are more accessible to particular antibodies than other. Overall I’m really happy with my results and I’m excited that I produced good data this summer.

Glaser-Hay Conjugation Results

After 7 weeks of research I had finally reached a point where I would be able to conduct experiments on the green fluorescent protein I had synthesized and collect results! I began working with the p-propargyloxyphenylalanine (pPrF) GFP as I had synthesized that successfully first. The unnatural amino acid pPrf is synthesized using the starting material tyrosine and propargyl bromide. Once the two are reacted, the hydrogen on the tyrosine side group oxygen is pushed off and the oxygen is now bonded to a propargyl group. The propargyl group, which contains a C-C triple bond, introduces a functionality which is not naturally present in amino acids and proteins. This allows us to conduct alkyne reactions with specificity. In order to understand the properties of the fluorophore, I set up an experiment where the UAA would be reacted with alkyne molecules. This unique reaction is known as the Glaser-Hay Reaction and allows us to create artificial polyynes. After the Glaser-Hay reactions were conducted with various alkyne molecules, emission spectra were collected for each of the reactions. We were able to see significant shifts in fluorescence peaks with the various molecules which meant that the weeks of work had paid off with some very interesting results!

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Goals with GFP with Unnatural Amino Acids

In my last post I discussed how my primary goal for the summer was to synthesize different types of GFP, all with some unnatural amino acid (UAA) inserted into the protein sequence. The UAAs that I inserted into GFP were 3-fluorotyrosine (3FY), ortho-nitrobenzyl tyrosine (ONBY), and p-propargyloxyphenylalanine (pPrF). The goal of inserting these UAAs into the protein is to observe how they modify the chemical properties of the GFP fluorophore. When analyzed with a spectrophotometer, wild-type GFP has a characteristic spectrum. Because the UAAs are inserted in the fluorophore region of the peptide, a shift in the fluorescent spectrum is apparent in GFP with UAAs incorporated. The next step in this project was analyze the protein itself using spectroscopy and conducting experiments on the protein to see if there is a shift in the fluorescence spectra.

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My Findings

Going into my research I had relatively little understanding of the economic realities of the Andes. As time progressed I made more and more hypotheses about the alto-Andean economy that I was itching to test, with all of them centered on the weaving industry that remains so central to rural Andean life and the women who perform the weaving. Among them were the following: [Read more…]