1-19-10: Sample Preparation

Today we have been working on preparing samples that we will run on our next western blott. On Thursday, the workers in our lab and I dissected mosquitos by seperating the head, thorax and abdomen. Dr. Nuss mixed different solutions that should extract different proteins from the mosquito parts into the solution. On our last western blott, we did not see any proteins from Aedes aegypti in our results, and Dr. Nuss wanted to try a couple of new extraction solutions that might cause the proteins we are trying to identify to go into our sample better. We took the solutions he prepared and centrifuged and dried them. Once they are all dried, we are going to resuspend them into a smaller amount of alcohol so that the protein content of the solution will be more concentrated than it was in the extraction solution. Then tomorrow we will begin running the samples on a gel. While the samples have been drying, I have been dissecting more mosquitos. Another postdoctorate associate in the lab is doing an experiment to isolate the mosquito insulin receptor (MIR) in the ovaries of the mosquito and then add it to synthesized ILP3 in order to test the affinity of ILP3 with the receptor. The dissection I did this morning was a little bit different; because the ovary needed to be isolated, we dissected by pulling the lower tip of the abdoment from the rest of the mosquito, which then caused the ovaries and intestinal tract to be pulled out of the mosquito abdomen. We then placed each pair of ovaries into a buffer so that he will be able to prepare a sample from the collections.

Last week when we photographed our results, I saved the pictures of our results onto my flashdrive in order to post them onto the blog, but I was unable to do so I think because the images are saved as .tif files. So, Dr. Nuss said that I would be able to label each of the lanes with which sample it was and change the tif file to a jpeg file with photoshop. So I installed photoshop onto one of our lab computers and am going to try to modify and post a pictures of last week's results.

This image is a photograph of the results of our first western blott from last week. Before adding the antibodies to the proteins on the paper, we cut the paper into three different segments so that we could test 3 different types of antibodies on our samples. The middle piece of paper was washed with a purified primary antibody while the other two side pieces of paper were washed with a raw primary antibody. The piece of paper washed with purified antibody did not show on the first photograph (30 second exposure) because there is no binding going on in the background. Antibodies are made by injecting the target proteins into an animal (in this case a goat), and in response, the goat's immune system makes antibodies. The raw primary antibodies include antibodies for other things that the goat encountered and are not seperated from our target antibody for ILP3. For that reason, the background of the paper washed with the raw primary antibodies are darker due to binding from the other antibodies in the raw solution.

Before we ran the gel, we mixed our samples into two different types of buffer (NuSep and Reducing). The reducing buffer functions to break the disulfide bonds that connect the A chain and B chain of the ILP. The NuSep does not do that and it is harder to compare the sizes of the bigger peptide with the A chain and B chain together with the ILP3 A chain standard and ILP3 B chain standard. The size is marked by the ladders that are in the middle of the three sheets of paper.

The lefthand segment of the paper was ILP3 A chain and B chain standards as well as Aedes aegypti head extractions (done via dissection, grinding and mixing with protein extraction solutions). If you look at the bands on the paper, all of the ILP3 chain standards showed up, which is good because Dr. Nuss had done a previous experiment in which they did not show. However, we did not see any bands for the Ae. aegypti heads. For that reason, today we prepared Ae. aegypti heads in different extraction solutions to run on the gel in hopes that we will be able to detect this ILP3.

On the segment of paper to the right we tested ILP134 standards and Anopholes stephensi heads. The ILP134 standard is made up of different segments of conserved ILP1, 3, and 4, and the antibodies should recognize any one of the three ILPs. Our lab has hypothosized that the function of ILPs 1, 3, and 4 is somewhat redundant and that is why we tested for all three of the ILPs. On this blott, we did get a band for the mosquito heads, but it was bigger than what was to be expected. If you compare the size to the standards, you can see the difference in the sizes.

Also, we tested the standards again by dotting the standards onto the nitrocellulose paper prior to the primary and secondary antibody washes. You can see this best on the segment of paper to the left in the bottom lefthand corner. We did this as a positive control because if there was anything wrong with the process before the antibody washes, then we would have seen the ILP3 dotted standards, but not the standards that went through electrophoresis on the gel and onto the paper. There is a small dot which is the B chain ILP3 standard. The different standards and bands look better or worse depending on the exposure time as well.

To finish the day, I am going to continue to help Animesh with ovary dissections.