Protocols, Training, and First Western Blot

My first few weeks here on campus were full of learning procedures and protocols so that I could start doing experiments. The first week and a half, I began by learning how to make plasmids, which are circular strands of DNA that replicate independently. Plasmids can be used to manipulate cells to see what happens when you over- or under-express certain proteins. For example, in my lab’s most recently published paper (Banks et al 2017), we found that when MK-STYX is overexpressed in primary neurons, it changes their morphology. Plasmid preparation consists of transforming the target DNA into E. coli cells and then isolating the DNA from the bacteria after letting them multiply. I then determined the concentration of the plasmids using the Nanodrop.

Over the second and third week, I was trained in tissue culture, the growth of cells from living tissue in an artificial medium. For the time being, I will primarily deal with rat pheochromocytoma (PC12) cells, so I learned how to grow and maintain PC12 cell cultures. I’ll start working with primary neurons later in the summer. I also started my first experiment, using 6 well plates and 60mL plates, at the end of the second week and finished it up during the third week. First, I transfected the cells in the 6 well plates with the appropriate plasmid, and then I stimulated some of the cells with nerve growth factor (NGF). After allowing the cells to incubate, I fixed the cells with formaldehyde and made slides with the coverslips from the wells. This preserves the cells and allows me to analyze them under the microscope. For the 60mL plates, I went through the same steps, but instead of fixing the cells for slides, I lysed and centrifuged the cells. I then removed the lysate from the tubes and used the Nanodrop to get the protein concentration. The protein lysates can then be used for a Western blot. Finally, I finished my first Western blot last Thursday, and I was also able to look at my slides under the fluorescent microscope! My next step is to repeat the experiment with the 6 well plates three times, so that’s what I’ll be doing this week and next week.