Thus far in our research, Dr. Krucher and I have made progress with determining whether or not certain resistance pathways in breast cancer cells remain inactive in response to reducing PNUTS levels or PNUTS Knockdown. One of the resistant pathways we have discovered as remaining inactive when PNUTS levels were reduced is AKT. In order to come to these results I would perform an experiment utilizing a series of laboratory techniques including but not limited to performing cell lysis protocols, protein concentration assays, western blotting, and running gel electrophoresis.
One of the challenges that I had with regard to our research was figuring out why some of the samples I had made were not easily visible or clean when it came time to view the Western blots via the Bio-Rad Imager. This problem lead to communication between Dr. Krucher and I in an attempt to determine why this was happening in my samples. After careful consideration and discussion with Dr. Krucher we came to the conclusion that the problem may have been a result of not always keeping the cell pellets on ice before performing the lysis protocol. This would potentially allow proteases within the cells to kill off the proteins that we want to isolate which would have accounted for the problems I had viewing the blots in the Bio-Rad Imager.
Through this project I have learned something new with each passing day. From learning laboratory techniques, to better understanding Cancer cells and learning more about the pathways that lead to their resistance, this research has allowed me to learn immensely and I am looking forward to continuing to learn throughout the duration of my time doing research with Dr. Krucher.