Blog Post #2 Discussion of my Progression in Research

Even though I have only been working about a semester in the research lab, I have learned an abundance of information and techniques in this short period of time. Personally, I feel I have made much progress already in the lab. Everything I partake in is on my own after I am shown what to do for the first time. This way I am able to learn more quickly and provide me the opportunity to learn for myself exactly how certain techniques are performed. More specifically, the techniques I have been taught and now mastered are Protein Extraction, Cell Lysis, Protein Concentration Assay, running of gels and the Western Blot, and 3-D culturing. 3-D culturing is difficult to master in the beginning due to the fact that you are working with Matrigel which is an extracellular-matrix material that changes between a more solid or liquefied substance based on the surrounding temperature. Therefore, culturing and feeding cells in such an environment must be taken with great care or then there will be no cells to work with for the experiment. Protein Extraction is a technique that dissolves Matrigel in order to release the cells and thus releasing the protein from them. Taking the samples from the Protein Extraction, a Cell Lysis procedure is performed to open up the cells to allow the proteins to be released followed by a Protein Assay, or a standard curve, to examine how much protein is truly in our sample as compared to the standards. After we determine how much protein is found in our sample, we can introduce the protein sample, a standard, and whatever other components are necessary into a gel and examine later in a Western Blot. Gel electrophoresis separates proteins by size and the Western Blot is used for protein analysis by identifying specific proteins with the use of monoclonal antibodies. Once the blot is completed, results of how much protein exhibited are apparent. These results allow me to conclude whether I received my expected results or if an error occurred due to my technical performance.

At this point in the semester, I feel I have obtained significant data even though my techniques and experiments have been mutually the same and repetitive each week. Repetition is not a bad thing though because through repetition I am perfecting my techniques and learning more and more with each recurring experiment. Especially with the research I am conducting in, I must make sure to keep a close eye on every detail and be very cautious and careful to not make a mistake due to the fact that these materials are super expensive. I have performed a few protein extraction, cell lysis, and protein assays, have cultured and fed MCF7 cells in 4 different slides over a span over several weeks, ran 2 gels and performed 2 western blots with varying times and examples per experiment. With each new experiment I run, my results become closer and closer to being more precise. At this point in time, I feel very comfortable with the proper techniques in order to carry out a full experiment with ease. I thoroughly enjoy everything I am working on and conducting in and each day I am more and more fascinated by my area of research. There is still so much to know and understand about breast cancer research and cancer in general therefore I strive to learn something new each day and continue to do so in the future.

Even though everything may seem controlled and understood with my research, many questions still arise here and there as I finish each experiment and move onto the next. A few weeks ago, my mentor, Nancy Krucher, mentioned to me that she is working on finding a new drug or procedure that can be introduced into 3-D cultures in order to induce apoptosis within the cells. PNUTS KD was recently found to not work in the 3-D cultures as it did in the 2-D cultures. Therefore, she must find a replacement product in order to continue on with our experiments with 3-D’s. In the meantime, I have been working on perfecting my techniques which I have successfully done. If I recall correctly, I do remember Dr. Krucher mentioning that she has an idea of something that might work but it does take several weeks to initiate. If her sample experiment seems to provide her with the expected results, then she will inform me of such product and allow me to begin working with it in conjunction with my own experiments. Overall, my general questions would be centered on what else I am able to learn at my stage in research. I am ecstatic to see what else is in store for me, what new product will indeed induce apoptosis in my breast cells, and to be able to continue working with Dr. Krucher.

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