There have been a lot of ups and downs during this past month conducting research. I have experienced the same imaging problem with Western Blots which I thought I had overcome. Unfortunately, I am still having trouble visualizing the protein of interest and am unsure where in the process something has been going wrong. As a check between steps in the long process of western blotting, we are able to stain the protein membrane after the protein has been transferred from the gel to the membrane. Normally we use a red solution called Ponceau S, but we have started to use a new protein membrane staining kit which should be more accurate and easier to wash off. The new kit has been very helpful in indicating the presence of protein and whether it is worth the time and reagents to continue with the rest of the procedure. This new kit has quite a few more steps than the original solution we used, but its all a matter of reading the protocol ahead of time. Much of lab work is following a procedure or replicating a technique. My mentor often recommends looking up the suggested instructions for various reagents and printing out the company’s protocol. We have also referenced many journals, reading for more insight into groups’ successes with a certain procedure. While reading journals, we noticed that two groups noted using a higher concentration of one reagent that was used in a solution for the western blot process. The researchers mentioned that increasing the concentration from something as small as 3% to 5% caused a decrease in the background color of the protein membrane and would allow for a clearer reading of the protein bands. Having increased the concentration, we did see better contrast between the protein membrane and the protein of interest.
This past month I tried to transfect cells for the first time. Transfection is a process of inserting genetic material into cells. In my case, I was injecting microRNA (miRNA) into neuronal cells. I had to continually reread the protocol and previous lab members’ lab notebooks for this experiment. I was trying to replicate the transfection done by a previous lab member, but in a different cell line and with different reagents. Since we were unsure of the degree of success for each reagent we also tested the dosage of reagent used. Acting as a no treatment control, I used the transfection reagents on the cells but did not include the miRNA. Then I had prepared a low dose and a high dose solution for the two different reagents and included the miRNA of interest. The cells were given some time to sit in the transfection solution before I lysed them and extracted the RNA. I then set up a miRNA quantitative polymerase chain reaction (qPCR) in order to quantify the amount of miRNA of interest in the different conditions. If there was an increase in the miRNA that we added to the cells compared to the cells that did not receive any miRNA then we know the transfection worked. We could also compare the two different reagents and the doses to see if one showed a relative increase in the amount of miRNA. With this preliminary experiment we were able to determine which reagent and method would be best for our cell line. Moving forward, we plan to transfect cells with the miRNA of interest and then also treat the cells with excess metal ions to better figure out the molecular pathway leading to the overexpression and hyperphosphorylation of Alzheimer’s disease related proteins.
So far I am still working on visualizing protein bands on the western blot. One of the earlier struggles I faced was that I had lower protein concentrations than we preferred for running a gel. Coupled with the fact that I was sometimes having trouble counting the number of cells for plating, my mentor checked the density of the cells on the plates and realized that I was plating at a lower density than what was recommended. I think overtime I was interpreting the density of the plate differently than what I should have been. For instance, when I thought the plate was ready to be split, my mentor showed me how it was still not confluent enough for splitting.
When splitting at a lower density than what is best for the cells, it takes longer for the cells to grow on the plate. This idea goes back to cell signaling lessons in Cell and Molecular Biology. If the cells aren’t in communication with each other then they will not continue to grow. Thankfully, we caught this mistake and can hopefully eliminate this error when considering why certain procedures have not been working out. In addition, since I needed to wait a week for the cells I was working with to grow up a little more, I was able to work with my research advisor more closely. We have a lot of various metal ions and small molecules we could treat the cells with and sometimes need to prioritize some over others, but I was able to help her extract protein and run a gel for quantifying protein because we were able to work as a team. We were testing these conditions in two different cell lines so the number of samples was doubled.
It has been a great experience working so closely with a professor in the lab. I have been able to ask questions small questions about a treatment condition or a step in the procedure which turn into greater conversations about the molecular mechanism in question and the many factors affecting it. So many enlightening conversations are in the air. I love stepping into other labs as well and hearing the different discussions about chemistry. It is also a neat way to learn more about people’s research as well as the different ways that their work may overlap with things we learned in classes or what we are working on in our lab. For instance, a friend of mine who is working in a biochemistry lab needed to extract protein with techniques we learned in our organic chemistry lab. I love the diversity of the research and the niche focus of the projects. I can’t wait to learn about everyone’s research projects at the poster session!