1-12-10: Mosquito feeding day...

Today Dr. Nuss and I started the second stage of the Western Blotting procedure which is called the immunoblot stage. During this stage, we took our dried nitrocellulose paper with the blots of our insulin like peptides and first blotted what are called standard ILPs onto the paper as a positive control. The standards are solutions that definitely contain ILPs and we blot them onto the nitrocellulose paper as a positive control because if the paper is somehow defective then the ILPs do not show up and we can know that if we don't see anything from our experiment that it is because of the paper and not another problem in the procedure. We then cut each piece of nitrocellulose paper into three segments according to which wash each sample needed to receive. We then soaked each of the pieces of nitrocellulose paper in TBS-T (triss buffer saline with tween 20) for a couple of minutes.

In the immunoblot procedure, there are two main washes that need to be performed: an antibody wash and a wash that contains a catalyst such as an enzyme. The first wash is the antibody wash because the antibodies in the liquid bind to the proteins on the membrane of the nitrocellulose paper. The second wash contains the catalyst and this portion of the procedure is done because the enzyme binds to the antibody and then a reaction occurs which produces a color and allows for the protein to be visible. We performed the first of the two washes by preparing a block solution from ECL and TBS-T and drained the TBS-T from the paper and poured our newly made block solution onto the paper and put it in a rocker in the cold room. After about two hours we took our papers from the coldroom and injected antibodies into each of the dishes containing the block solution and paper. Now, the samples with the antibodies are on the rocker in the cold room and we will finally get our results tomorrow.

Then we made up some new TBS buffer by mixing NACL, HCL, Tris base and deionized water. It took a while to make the buffer because the HCL that we were using was highly concentrated and dangerous to handle. We only handled a small amount at a time (about 10 mL) and then safely disposed of the extra that we had left over.

Then I labeled 30 small tubes because tomorrow wer are going to alloquat a large amount of an antibody solution into the smaller tubes so we can put the solution into the -80 degree refrigerator so that bacteria does not begin to grow in the solution. Dr. Nuss explained that it is important to put the solution into small amounts because if you freeze and thaw a solution containing proteins several times then the proteins begin to degrade.

Before I went to lunch, I went into the mosquito room because another person working in our lab needed to bloodfeed the mosquitos so that they would mature and be able to produce eggs. He had anethesized a rat and put it on a screen for the mosquitos to feed on it and it was kind of neat to watch. He is doing an experiment that involves injecting different solutions into the mosquito that will hopefully stop the genes controlling insulin like peptides from being expressed. He let me inject one of the mosquitos using a small needle and syringe and it was really tedious, but it was kind of cool because first he froze them so they could pick them up and handle them, injected them and then put them in a container and right after the injection they would start flying around like nothing happened.

Tomorrow we will get our results and I think start a second western blot.