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.

A new test with an unexpected result

Since my ChIP is going slowly, I decided to use a genetic approach to determine how well htt activated inappropriate genes. I used an artificial plasmid which included a reporter gene that could only be activated by htt binding to the promotor. I then did an ONPG assay to measure how well htt bound to this promotor. Surprisingly, I found that 25Q was better at activating this promoter than 97Q. This surprised me since other evidence from my lab and other labs shows the opposite, therefore I will need to figure out what is going on in this experiment and why.

Changes to my experiment

At the beginning of the summer I figured out how to do the ChIP protocol. However, I wasn’t getting the results I expected. So I started to change conditions to try to find something that worked. First I got new antibodies that are designed against htt which should work better than the indirect antibodies I was using before. I also started to use new htt constructions which should hopefully interact with Slx5 better than my old versions. Hopefully, when I do a ChIP with these changes it will work better.

The summer of ChIP

Hi,

I’ve had a busy summer. All summer I have been working on my Chromatin Immunoprecipitation (ChIP) assay with the hope of showing that the Huntington protein can bind to DNA. After my second try I think I got it to work. However, the results were inconclusive since the no antibody smaple had the same fragment amplification as the sample with htt. Therefore, I think the antibody I used was not working, so I ordered a new anti-htt antibody which will hopefully allow me to successfully complete the assay. If I’m lucky by next week I should have results!

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