Stop! You’re Stressing Me Out.

As in most labs, each day at the Silva Lab holds something different. There are routine tasks that must get done, like prepping cells, making media, and autoclaving flasks. There are frustrating tasks that must be dealt with, like troubleshooting protocols, testing new antibodies, or working with very old computers. And finally, there are exciting tasks that you hope for, like analyzing the interesting results from your latest experiment and planning what you should do next.

I’ll generally plan out my entire week ahead of time based on my progress/results from the previous one. Recently, my weeks have begun to look a little different as I’m learning some very cool and exciting protocols, including Myc tagging proteins through PCR and staining cells through microscopy! Despite these new additions to my week, one experiment continues to be a part of it. As my lab is focused on the cellular response to oxidative stress, the most common experiment I perform involves stressing out yeast cells!

The standard oxidation experiment involves several stages. First, I must inoculate all of the necessary strains of yeast cells in my desired media, allowing them to grow overnight. Next, I’ll dilute them in new flasks. I do this in order for the cells to be at the correct growth phase, specifically log phase, by the time I will be doing the experiment the following day. Next, I will subject the cells to oxidative stress by exposing them to low concentrations of hydrogen peroxide. Once treatment has been completed, I’ll collect cell pellets from each sample. The next step is to lyse (break) these cells open in order to extract their lysate. After that, I perform a Bradford protein assay which is a neat way of measuring the concentration of proteins in your lysate samples. This assay allows me to normalize the concentration of proteins across my samples for more accurate results later on. Treating, lysing, and Bradford generally take about a day. Thus, the following day is usually when I’ll run a western blot, which is a method used to detect and quantify the presence of specific proteins in a sample. This is done through antibodies. Once I run the gels, transfer the proteins from the gels to my membranes, incubate the membranes in the antibodies for the proteins I’m interested in, incubate in secondary antibody, and develop via film, I will go over results with my mentor and hatch a plan for what I should repeat, change, or test next!  

Image from: https://www.arborassays.com/products/by-research-area/oxidative-stress/

In between cell lysis or antibody incubation, I’ll make sure my lab notebook is up to date and (of course) read the literature. Each week I’ll have papers to read for BSURF’s journal club, for my lab’s journal club, and for my project. While oxidation experiments are yielding interesting results, I am most looking forward to mastering the new protocols and analyzing the data that comes from them!

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