As we approach the end of the semester, our research is beginning to prosper. The time has come where we are gathering all of our information and beginning to write our documents to be published. Dr. Krucher is leaving for San Diego in the next few weeks to present our information to The American Association for Cancer Research which is very exciting for us. We have overcome the road blocks that we encountered during the middle of the semester and can now continue to discover new qualities from these breast cancer cells that we have been manipulating.
During the semester, Dr. Krucher and I were having a hard time extracting the protein from our 3D breast cancer cells. It was difficult to separate the protein because the material that the cells are growing in is also made of an extreme amount of protein, leaving it impossible for us to measure how much protein we were able to obtain, if any. We attempted this procedure a number of different ways until finally, we had a measurable number! This was very exciting, but it gets even better. We ran our Western Blots and found that they came out fantastic! The results were very clear and much more precise and accurate than we thought they were going to turn out.
We were also able to conduct Immunoflouresence on our cells, which doesn’t require any extraction of protein. It actually only works when cells are intact. At first we found through these experiments that our transfections were not working. Transfections are the addition of certain nucleic acids to another cell, which changes the gene expression of that cell. We have worked out the problem with our transfections and have now begun to see our results. I really enjoy these experiments. We do not have the powerful microscope that is necessary for us to analyze our results for these experiments, but another teacher, Dr. Horne does. He works this machine that is so big that it is almost hard to believe it’s a microscope, and displays our results on a computer that is connected to the microscope. Now that we have worked through obstacles and mistakes, we are able to continue on the track that we had planned before those problems.
I am very much looking forward to what is to come. The vibe in our lab is completely different now that we are coming so close to combining all of our information for a paper to be published. We are all working hard as ever and are staying enthusiastic and determined. This experience has been one of the best experiences of my life. I have completely fallen in love with doing research and definitely want to continue it in a life-long matter. I hope to work in the research lab at the next school I want to go to, SUNY Optometry. I simply couldn’t imagine myself not doing research. Like I said in the first blog I wrote – Once you’re in it, you’re hooked.
As 2014 is beginning, Dr. Krucher and I have found ourselves faced with the challenge of beginning our 3D cell cultures. This is a struggle for both of us, as it is a new technique to our laboratory. We have taken the time to put aside our Western Blots and Immunoprecipiations in order to become experts at taking care of our 3D cells. In this case, we have cell lines MCF10A, 231, Hs578T, and SKUT1 growing in 3D cell culture.
Taking care of 3D cells is not unlike taking care of your children. You have to feed, nurture, and change them while carefully watching them grow up. This plating technique is different in such a way that the cells are able to mimic how they would behave in their normal environment, interacting with all three dimensions around them. Contrary to 2D cell culture, these cells are plated with a material called Matrigel. This substance is a mix of Collagen and Laminin which are the substances that let the cells recognize as their “natural habitat”.
Dr. Krucher and I have started to attempt extracting the protein from these cells. Extracting the protein is how we conduct most of our experiments in the laboratory. Unfortunately, the various techniques that we have used already have not been successful. Although we are not able to extract the contents of these cells at the moment, we are able to tag different proteins while the cells are still intact. This experiment is called immunofluorescence.
The process of immunorfluorescence is done through the use of antibodies. Protein-specific antibodies are used to tag whichever protein you are interested in. Then, a fluorescent secondary antibody recognizes and binds to the primary antibody, which gives the experimenter the ability to visualize the target protein via microscope. This technique is useful for localization of certain proteins in the cell, which could be important in our research.
Once our trouble with extracting protein is solved, we will be able to continue doing Western Blots and Coimmunoprecipitations to further study the association of Rb with protein Bax as suggested in prior blogs. I am very thankful for this opportunity to do research in such a wonderful setting and look forward to continuing my research with Dr. Krucher.
Breast cancer is the most common form of cancer women are afflicted with, and about one in every eight women will be diagnosed with it in their lifetime. It can affect anybody from your neighbor, to your teacher, and even your own mother. It is for these people that we love so dearly that many scientists are searching for a cure. Breast cancer is a complex disease, but that is my favorite part of our research. My faculty partner and I are looking to explore the significance in the interaction between a tumor suppressor called Retinoblastoma (Rb), and the pro-apoptotic protein Bax in cell culture systems and in three dimensional cultures of breast cancer cells.
Rb is a tumor suppressor protein which prevents excessive proliferation in cells depending on its state of phosphorylation. Phosphorylation is a chemical modification of the protein that turns it on and off. When Rb is highly modified by phosphorylation, cancers such as breast, ovarian, and colon develop. Our research focuses on the phosphorylation of Rb, which has 15 potential sites. In previous work, it has been shown that modification of some sites gives cells the ability to evade apoptosis (cell death). Therefore Rb controls life and death in the cell. This project will test the hypothesis that certain phosphorylation sites regulate Rb binding to other proteins, such as the pro-apoptotic protein Bax. Association of Bax with Rb is a new finding that we plan to further investigate in these studies.
In order to find out what sites of Rb phosphorylation are involved in association with Bax, we will use plasmids that express mutant Rb in breast cancer cells. Following this we will determine the association between Rb and Bax. These studies require a great deal of laboratory work. Cell stocks must be grown under aseptic conditions, in complex media, for weeks at a time. Experiments take 4-5 days, starting with setting up on Mondays or Tuesdays followed by analysis of the experiments on Thursdays or Fridays. To analyze, we use laboratory procedures such as Immunoprecipitation and Western blotting. Luckily, I learned these procedures this past summer while training in Dr. Krucher’s lab. These experiments determine the absence or presence of proteins, as well as binding of specific proteins. Because the experiments are complex and challenging, and one or other small steps often go wrong, we need to troubleshoot each week until we obtain results that we believe are correct and are reproducible. Therefore it will likely take almost the full academic year to find our answer.
For the sake of our loved ones, it is vital for people like my faculty partner and I to be dedicated to our research. It takes an enormous amount of determination and brain power to conduct these experiments, but once you’re in it – you’re hooked. I believe we can make a difference in the future of breast cancer and hopefully through these experiments, all of us will be able to see it unfold.