We started off our week with a congratulatory acai bowl trip to celebrate Preston’s acceptance into a training grant program. Acai bowls in California top Playa Bowls (no question about it). From what I can tell, its a pretty huge honor to be recognized by this grant, but he’s really humble about it. On Monday, Preston and I decided that testing antibodies that have never been tested on prostate epithelial cells before would be a good objective for my first Western blot on my own. We needed to probe for ASCT2, a glutamine transporter, and GLS in order to determine if their corresponding antibodies are functional. Antibodies are crucial for Western blots because they bind to the protein of interest (POI), allowing for us to qualify its expression after imaging. As such, Preston wanted to make sure they worked by probing for ASCT2 and GLS on three different cell lines. Cell lines are commercially purchased human cells that have been immortalized (modified to grow indefinitely) by telomerase overexpression, which adds more telomeres to the end of chromosomes. We tested three cell lines, RWPE, DU145, and VCAP. We wanted to analyze LNCAP as well, but we did not have enough of that protein sample. Before beginning, we checked online to determine the sizes of ASCT2 and GLS proteins. Then we performed a BCA Assay, which evaluates the concentration of the protein lysates by quantifying its absorbance after the addition of a reagent that contains dye that interacts with protein and changes the solution's color from green to purple (thanks, copper ions). We had to compare them to a standard, so we made nine tubes of linearly increasing concentration through serial dilutions. Technology graphed absorbance as a function of concentration, spit out an r squared value, and gave us a nice equation (y=0.0005x +0.0918). The machine gave us the absorbance, so we did some algebra to find the concentration of protein lysate in the cell line. From there, I calculated the volumes of the constituent components for what we load in the gel using a set of complex guidelines. If you’d like, here are some numbers:
Running a gel with protein is much different than the good old DNA gel electrophoresis I remember from sophomore year biotech. For one, it stands vertically, not horizontally, which makes it really difficult to load the gels with my uncontrollably shaky hands. I did it though, with the moral support of Preston, and it didn't turn out too bad. I’ll fast forward a little bit to where we imaged our membrane on the Typhoon 9000. Looks good, barely any bubbles: 
In other news, I found my PI in line at a vegan restaurant I wanted to try. I got the beyond burger, some plant based patty that is so similar to meet it even oozes beet juice when squeezed. It scared me a little bit but it was pretty cool.
Also, I learned that ATP synthase can run in reverse, messing up the membrane potential in luminal cells especially, leading to a whole mess that likely contributes to rapid luminal cell death in organoid culture. Preston wants to look a little more into that--he gave me the whole spiel on luminal cell metabolism (well, I suppose in actuality a very simple version of it. But to me, quite complex). Anyway, now I have a lot of questions on that. And I’ve got quite a few things on my mind with regard to reactive oxygen species. Preston is pushing me to talk to Dr. Goldstein about looking into cytokine deprivation and ROS levels after I mentioned to him how I read about it when I studied the impact of an inflammatory microenvironment on DNA damage. Maybe I’ll mention it if the topic ever happens to come up at a lab lunch… For now, I'm reading papers that explore the link between ROS levels and chemokines just in case I ever decide to discuss it.
For the weekend, I headed down south near Newport Beach. It is expensive to uber that far so I tried to join UCLA rideshare groups on facebook. That didn’t work:( Ended up catching a ride with this guy and his girlfriend who I met at his frat house. Interesting situation for me. His girlfriend ended up working in the lab next door to mine! Small, small world.
Comments
Post a Comment