1-13-10: Results today!

First, I am going to lay out the Western Blott procedure because I do not think I have fully understood it until today and could completely explain it in a simple way:
1. First, you prepare your protein samples (our samples are controls that definitely contain insulin like peptides and mosquito body parts that we don't know if they contain insulin like peptides or not).
2. Load the samples into a gel and electrophorese in order to seperate the proteins into different sizes. Proteins with a higher molecular weight appear higher on the gel and proteins with a lower molecular weight appear lower on the gel. We also load markers which indicate the size of the proteins that show up in our sample.
3. Transfer the proteins from the gel to nitrocellulose paper. This is done by placing the paper next to the gel and running another electric current through the gel in order to pull the negatively charged proteins from the gel to the positive pole and onto the nitrocellulose paper.
4. When the proteins are on the nitrocellulose paper, they are not visible. To make them visible, we add antibodies that are specific to bind with the ILPs.
5. We then add secondary antibodies to the proteins and the antibodies that is specific to bind to the antibodies. When the secondary antibodies bind to the antibodies, a reaction occurs, a color is produced, and you know whether or not ILPs are present in the mosquito heads.

Today, Dr. Nuss and I finished our first Western Blott. We left the protein samples on the nitrocellulose paper to soak in the primary anitbodies overnight last night. When we came in this morning, we did 3 washes for twenty minutes each with the TBS-T (tris saline buffer with tween 20). Then, we added the secondary antibody to our proteins and allowed that to soak for about four hours. And then we were able to photograph our samples and view our results. Before I started working this week, Dr. Nuss ran a Western Blott with chain A standard, chain B standard, mosquito heads, thoraxes and abdomins. The bioactive form of insulin is made up of an A chain and B chain connected by a disulfide bonds. The chain A and chain B standards are the segments of insulin, and should definitely show up on the Western Blott. One of the first times Dr. Nuss ran the procedure, he ran it with these standards, but for some reason the standards did not show up in the results when he ran it. The Western Blott we have been running the past three days included chain A standards, chain B standards, and mosquito heads. In our results, we were able to see the standards, so the procedure worked, but there were no bands for the mosquito heads. Dr. Nuss said that he thought it may have been a problem when making the mosquito extract.

We also started our second Western Blott by preparing our samples and running them on a gel. Then we transferred the proteins from the gel to the nitrocellulose paper and the paper is on the lab counter drying.

Tomorrow I am going to finish the second Western Blott that we started today and dissect some mosquitos because we are going to increase the number of mosquito heads in our next Western Blott to see if increasing the amount of mosquito juice will give us a band on our next Western Blott.

I also spoke with my dad (who is a gastroenterologist) and he has arranged for me to shadow one of his friends, Dr. Manning - a family practicioner, this Friday.