The goal of the research I have been doing this year is to activate the tumor suppressor Rb, in breast cancer cells. To do that we activate an enzyme called PP1, which activates Rb, by a gene silencing technique called PNUTS knockdown. So far in this research I have learned many of the techniques necessary for setting up PNUTS Knockdown. I have learned how to efficiently count and plate MCF7 breast cancer cells, as well as learned how to trypsinize cells off of plates in order to remove the cancer cells and centrifuge them to collect them.
Throughout the semester I have also learned more about the intricacies of the PNUTS KD For example, I learned that using specific antibodies we tag the proteins that are on the nitrocellulose paper and after viewing them via specific computer software (Bio-rad image software) we determine whether or not PNUTS was knocked down by comparing a portion of cancer cells that didn’t receive any treatment (controls) that knocked down PNUTS to a portion of cancer cells that did have PNUTS knocked down and if the software shows the band where PNUTS is to be lighter or less prominent then we know the knockdown was a success. Furthermore, we also look to see if the band where Actin is, is more prominent or less prominent or the same. Ideally it is the same, meaning the same amount of protein was loaded in each cell. Therefore, expression of actin serves as our loading-control.
So far we don’t have any confirmed data because the results we do have need to be replicated several times to have confidence in the accuracy of the results. However, we do have general results. For example, so far the experiments that have shown promising results regard the phosphorylation of AKT detected by the antibody AKT-473. The way we know this antibody has shown promising results is by viewing the band on the nitrocellulose paper as mentioned before and determining whether or not the band is more prominent with the treatment or less prominent and in the experiments we have carried out so far the bands for this antibody have been less prominent after the PNUTS KD and this experiment will be carried out again a number of times to ensure the results are accurate.