Week 2 of my rotation was dedicated to tweaking the experiments that I performed during Week 1. I repeated my western blots a few times. First, I used the original protein lysates in larger amounts, thinking that perhaps my first blots failed to detect the protein of interest because it's not very abundant. I still didn't see anything, though. So, on the advice of the postdoc who has been training me, I made some fresh protein lysates from wild-type mice. I had never done that before, so it was a good chance to learn a new technique. I had to look up some diagrams on the internet to figure out where, exactly, a mouse's pancreas is, but I think I did a good job in the end. I've always had pretty good hands for dissections, and while it may sound morbid, I think it's interesting to take things apart.
We thought the fresh samples might be helpful in case our protein of interest was being degraded in some way over time. However, when I ran the new samples on a gel the very next day, I still didn't see very good bands when I probed with our antibodies for this protein. One of the blots had pretty high background, the other showed almost no signal. I washed the high-background blot over the weekend and I'm re-probing it today to see if I get a better signal to noise ratio this time, but I'm not feeling particularly optimistic. Oh well -- that's why there's a "re" in "research!" I'll keep trying new things to find this wily protein.
Our plan for this week is to purify the protein from my samples by immunoprecipitation, then run a new gel and probe for it there. I've never done an IP before, either, so I'm excited to do more new things. And even though my experiments haven't been going so well, I still derive some satisfaction from methodically planning how to improve things with the next set of experiments. And, of course, I appreciate those experiments that give me blocks of free time for writing blog posts! I should probably finish this one up now, though, and get back to work on that essay for my seminar. Tune in next week to see if my IP worked!