Since mid-June, I’ve been advancing my research project within the walls of the Carlton lab located at NYU’s Center for Genomics and Systems Biology. In adjusting the protocols for western blots and immunofluorescence experiments as part of my project (see previous blog post for background), I’ve produced some excitingly successful results, though I’ve also hit some barriers to optimization in recent days. However, I still remain hopeful for more results in the coming weeks!
During my first week, I began by thawing some of our Trichomonas vaginalis cultures in order to grow them before use in experiments. We are using a strain called G3, which is a commonly used reference strain which grows very easily. Below, you can see a photo of these guys under the microscope, which I use to count the number of parasites daily.
In the the last few weeks, I received the first results from each experiment after a bit of tweaking to the protocols. The image below shows the process for a western blot experiment, which I’ve been focusing the majority of my time on. Western blotting is a common experimental method used to identify specific proteins. Western blots require us to take a bunch of cells, break them open so we get a mixture of proteins, and then run these proteins on a gel so they separate by size. Then, we can transfer these proteins to a more stable paper-like membrane, and use different antibodies to identify our specific proteins of interests. Unfortunately, because it is a very long and complicated experiment, there are many places that it can go wrong. Temperature, timing, light, and other (annoyingly) specific conditions all can make a huge difference! However, after tweaking several steps, I was able to produce successful results for our control protein, Actin. That is to say, a western blot using antibodies for actin (which is a very abundant protein in Trichomonas vaginalis) showed fluorescent bands in the expected location. I won’t be posting photos of experimental results on the blog, but I can say that though this may seem like a small step, it is a good sign that I’m going in the right direction.
Unfortunately, though I’ve had success with the initial stages, I am having difficulties optimizing our blot for recognizing argonaute, our protein of interest. Despite trying multiple conditions and concentrations of antibodies, imaged blots have consistently been lacking the visible band that we would expect, suggesting that there may be issues with the antibodies themselves. For the remaining two weeks in lab, I will be continuing to try a few more experimental conditions and solicit other opinions and expertise, as well as continue with my other immunofluorescence experiments which I’ll speak more on in my next blog post.