Time to Analyze

After work began back on campus this summer, I began pouring through both the data from my surveys and the responses from the Comité de Salud’s health census that they had given to me while in country.

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Writing an Article

As a summary for my summer research, I wrote an article to submit to a research journal. The writing process did not take as long as I expected because I was so familiar with the information. I did have to spend quite a few days deciding which data to include. I couldn’t publish a graph of every single species change, so I had to decide which graph summarized each individual process the best. The most difficult part was reading through all the requirements of the particular journal I chose and making sure my paper, figures, and references were all in the correct format. The first journal I selected got back to me within 24 hours and said that the subject was not relevant to their target audience, but I reworked the formatting and submitted it to a different, less specific journal. This time, it took about 10 days for an editor to get back to me. This second journal accepted the article for peer review, and now I am waiting to hear back from them again with a final word on whether it’s been accepted as well as any corrections from the peer reviewer. I am so excited to share my research with a wider audience!

Week X and Final Goals

I apologize for the lateness of this final summery; tying up the scope of my research has been tricky and required a long period of meditation.

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The Highs are worth the Lows

My first Western Blot ever. I am thinking of framing it. I was so proud of myself. I did everything from scratch. I know it may seem simple and everyone in the biology world might know how to do it, but it was my first and it was a big thing for me. I casted the stacking and the resolving gel. Again I have learned the WHY of everything. I now know the importance of the pH difference between the stalking and the resolving gel and the importance of the amount of polyacrylamide one adds (depends on the size of the protein of interest). If you study a big protein then you add less polyacrylamide in order for the big proteins to run faster down the gel. The Ammonia Persulphate Solution (APS) added to preparation of gel acts as an initiator and the TEMED as a catalyst so you add those two components last in order to begin polymerization of your gel (not before or it becomes a solid before you can cast it). I heated my samples with loading buffer which contained SDS and loaded it with marker and previously collected samples. I covered everything with running buffer at working conditions (1x this term is also new to me). Then transferred my proteins from the gel to a membrane via current again. Once my proteins were in the membrane, I blocked the membrane with milk, yeah milk, inorder to prevent unspecific binding of the primary antibody and then added the diluted rabbit antibody overnight which was specific to protein of interest which was CHIP in this case. You have to pay attention to where your proteins are at all times and having a marker with protein sizes helps. CHIP was around 35kDa. Then I had to come in the next morning to do washes and place secondary antibody which was anti-rabbit. This secondary antibody is special in that it has Horse Radish Peroxidase. It is used to locate the protein of interest since it is an enzyme that provides a detectable signal. In order to obtain this signal you have to go down to the dark room just like photography to develop film. In the dark room you provide the enzyme with substrate and then it turns it into product that can be detected with the film. It is the coolest thing. I have also used another technique where you can scan the membrane and it detects fluorescence but here you have to use a different secondary antibody, I do not know the specifics just yet, but it’s easily done which I like aswell.
I am telling you all this not because I want to bore you, but to show you how much I have learned… I do not want to simply to tell you that I have learned a lot… I want you to believe me! The more I think about it, the more grateful I am for this experience. It was extremely hands on. It showed me to think and to practice. Yeah it was a lot of mistakes. So many, but that’s what really makes me appreciate it. I am so happy. This has totally been worth it, every minute.

Wrapping Up

In concluding our research, we didn’t see much of a difference in AChE staining between the two hemispheres in the three rat brains from Hampton-Sydney. We are going to try the stain one more time, because it was a bit faint, and just to make sure we’re not missing anything. We will use new tissue sections for the second round of AChE and Parvalbumin staining within the next couple of weeks. With so many steps in each staining procedure, one small miscalculation can throw off results. However, it could just be that the results are accurate, and that there is no difference in AChE staining with Anti-Chat saporin, which would refute our hypothesis for the pilot study.