01-11-10: First stage of Western Blotting technique

The main project that Dr. Nuss and I will be working on is where insulin like peptides are located in the body of the mosquito. At the meeting last Thursday in the Holcomb room the question of whether the insulin like peptides (ILPs) in the mosquito are produced by the mosquito or if they are from the insulin taken up in the blood meal. I asked Dr. Nuss this question and he said that there are several ILPs, ILPs 1-5, and that they are all endogenous, meaning that they are all produced by the mosquito. Dr. Nuss also explained that part of the mosquito's immune defense system is to release free radicals in order to kill outside pathogens that may be harmful to the mosquito. One of the functions of insulin like peptides is to suppress the mechanisms that control the free radicals. So, when high amounts of insulin are present, free radical production is higher because there are fewer active mechanisms to control the put out of the radicals. When the free radicals are released within the mosquito more often, there is more damage done to the mosquito, and its lifespan is shortened. Although Dr. Nuss and I are not working on this aspect of the research, the lab is doing research to find out if a blood meal containing high amounts of insulin will ultimately shorten the life of the mosquito because of these mechanics. Dr. Nuss and I are working to learn more about insulin like peptides in the mosquito species Anopholes stehpensi and we are starting by doing experiments to find out where insulin like peptides are located in the body of the mosquito.

One type of experiment that we will be doing to identify ILPs in the mosquito is a technique called Western Blotting. Last week, before we were able to start experimenting today, Dr. Nuss had to do some preparations to run the Western blot. One thing that he did before we could start the experiment today was to disect about 500 mosquitos by seperating the heads from the body, grinding them up, centrifuging them and collecting the supernatant to run for the Western Blot. We are doing this experiment in order to identify whether or not the ILPs are located in the head of the mosquito. Today, there were three main parts of the first stage that we ran: preparing the protein samples, running the samples on a gel, and then transferring the proteins from the gel to nitrocellulose paper. We had 16 samples to run on our gel, and they were all either samples of mosquito heads, chain A of insulin or chain B of insulin. Insulin is a hormone made of of chains B, C, and A, but chain C is cleaved out of the active form of insulin and chains B and A are connected by disulfide bonds. So the samples with chain A and chain B are just insulin. We mixed the chains B and A, and the mosquito heads with different buffers (sample buffer and reducing buffer - which serves to bind with proteins that may be "sticky"and bind with anything, such as the proteins we are trying to detect) in order to make our 16 samples. Once our samples were made, we loaded them into a gel and did electrophoresis for about 3 and 1/2 hours. During that time I went to part of a seminar on termites and how they could be a valuable energy source because they could be used as a high calorie food or to produce high amounts of methane gas. Then I went to lunch with the program director, Dr. Marianne Robinette. After that, Dr. Nuss showed me how to set up the next part of the Western blot. During the remainder of the time to wait for the gel, I read a primary literature article that relates to the research we are doing. Once the gel was ready, Dr. Nuss showed me how to prepare the Western Blot "sandwich". We prepared two pieces of nitrocellulose paper on two sponges and then put our gel in between the two pieces of paper and into a machine to transfer the proteins on our gel to the nitrocellulose paper. The transfer to the nitrocellulose paper took one hour and I read part of another literature article. Then we were able to look at the nitrocellulose paper and our markers were visible. Tomorrow we will need to do a couple of washes over the paper that will cause the proteins to change color and be visible in order to see if they transferred to the paper. That was all we did for today and tomorrow we will be finishing up the Western blot.