Friday, January 9, 2009

Rotation #2 Diary: Week 1

As a first year PhD student, I'm currently working on one of my three required laboratory rotations. The purpose of a rotation is to "test drive" a potential mentor's lab before choosing where to do one's dissertation research. It's also a good way to pick up useful new techniques from a lab, even if you don't end up joining that group. 

I did my first rotation over the summer and was there full time. For the first week or two, I was pretty helpless and needed help from the postdoc who was assigned to train me every time I did anything. I also spent a lot of time reading papers and trying to get a sense of what the lab studied, because I couldn't start on some experiments for a while (such is the way of mouse-based research... sometimes the breeding schedules don't cooperate). After that time, though, I really got into the swing of things and really enjoyed my project. I got some cool data. My mentor wrote me a good evaluation. So, I came out of that experience having learned a lot and feeling like I'd be happy joining that group later on, but also excited to see what was in store for my next rotation.

I'm on my second rotation now, and although I've only put in one week, I'm already really impressed by the usefulness of rotations as a strategy for choosing a lab. Noting the similarities and the differences between the two places I've worked so far has really helped me to think about what I want out of my graduate career, what kind of experiments I like to do, and so on. I haven't decided where I want to do my third (and possibly last; I have the option to do another one) rotation yet; I'm going to wait until I'm done or nearly done with my second to see if I'm inspired to go in any particular direction by my current project. I thought it might be useful to keep a rotation diary to help keep track of what I'm doing so I can look back on it later. This should help me reflect on this rotation experience as I look toward my next one, and also as I look back on all of them when it's time to choose a lab to join. Plus, I can keep track of what I've been up to, what techniques I've learned, and so on so that when I have to write my rotation summary I don't need to pore over a chemical-stained lab notebook (though I have one of those, too, which goes into more tedious and potentially sensitive detail).

So, my current project. This lab uses a lot of molecular biology techniques, some of which were familiar to me. This week was mostly dedicated to western blots, which I've done before, so I was able to hit the ground running and do experiments basically on my own for much of the week after minimal instruction. I didn't really get any useful data, but... that's not necessarily what rotations are for. 

I'm doing some experiments to characterize a protein that I won't name, since the lab hasn't published anything about it yet (though others have). I was given a bunch of samples derived from rat tissue and told to see if I could detect the protein via western blot. I learned how to calculate the protein concentrations of the samples by doing a BCA protein assay, which was cool. I got to make pretty color-changing solutions in a 96-well plate, and use the plate reader! After calculating the protein concentrations, I was able to come up with a fairly accurate loading scheme for my gel. In the lab where I learned to do westerns, we'd run new samples on a gel and do a Coomassie stain to assess relative protein levels basically by eyeballing it, then adjust the loading using our best guess. Doing the protein assay is a lot more precise. So I loaded and ran my gels and probed them with the antibody for my protein of interest, and... it didn't work. I didn't get any bands. So then I washed the blots and reprobed for actin as a control, and the actin was all over the place. 

Whaaa? But my loading was determined by such an accurate method! So, then I learned that crazy actin level differences happen when you're using samples from different tissues, even if they have the same total protein concentration. I'd never compared such disparate tissues in one blot before. My previous western experiments usually used cell extracts that were all made from the same cell line and thus good controls for each other. Thinking about it now, it makes sense to me that say, skeletal muscle would have way more actin than ovary tissue, but I'm just used to seeing actin as a loading control so I freaked out when I saw the wildly different actin levels on my blot. The purpose of this experiment is to see whether my protein of interest is found in any of these tissues (since I'm in a neuro program, we of course hope to find it in the brain, but it could be elsewhere, too) and I guess we'll just trust the BCA assay (confirmed by eyeballing the blot after staining with Ponceau Red) to show that the loading is even. Of course, this is all moot for now, since I couldn't detect the protein of interest at all, in any of my samples. 

Our current theory is that this protein is finicky and easily degraded. The postdoc who's helping me with my rotation says that she's successfully gotten results from this antibody, but only on really fresh samples from cultured cells or brain lysates. The tissue extracts I used were made in 2005 and have been stored in the freezer ever since. So, next week we'll dissect some mice and make fresh samples to see if my antibody can detect anything then. I'll also try running a new gel using the old samples with double the loading, in case the problem is just that this protein exists at very low levels. Although it's frustrating when experiments don't work, I like having some ideas about how to troubleshoot. These experiments seem a little more methodical than some of the stuff I've worked on previously (neuroanatomical studies in different mouse models of disease), a little easier to tweak when things go wrong, so I'm feeling optimistic that even if I don't get the result I'm looking for, I'll learn something useful by troubleshooting and testing things. It's been cool meeting with the postdoc and exchanging ideas on what we can try next. 

I'm happy that I started this rotation early. My classes don't begin until late next week, so I've had some time to spend entire days in the lab. I think this project should lend itself well to my class schedule, though, since I can easily come in before class, load a gel, leave it running while I go to lecture, and come back after class to transfer/block/probe and so on. On days when I don't have class, I can use the down time during westerns to study, or to work on other projects related to the rotation. 

Anyhow, I will try to keep up this diary as the semester goes on.

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