It's weird. The past two weeks I've had a renewed sense of "I need to get my shit together and get the hell out of grad school." To the end that I'm busting my ass to get science done, asked a friend for a copy of his thesis (so I can start thinking about writing my own), and told a committee member that my post-Ph.D. plans do not include doing more scientific research.
More specifically:
I hate optimizing experimental protocols. This is why I have "bad hands" in lab. I'm great at coming up with project ideas, great at figuring out how to approach them experimentally, but when it comes to tweaking the protocols to make them work, I just don't have the patience. It's hard for me to repeat an experiment 8 times over, slightly different each time, to see which way works best. I just want someone to hand me a set of instructions to follow that WORKS. On a side note, this is probably why I'm better at baking then cooking.
But I digress. The point is, I hate optimizing experiments. Yet, I've just mapped out two weeks worth of doing just that. Because once this protocol is ironed out, I will be a hell of a lot closer to being done.
What am I doing, exactly? Trying to purify a protein out of cells, along with any other proteins that happen to be bound to it, and then identify those other proteins. To do this, I've "tagged" this protein with an amino acid sequence that binds to IgG, which can be attached to tiny beads. So you take a bunch of cells, burst (lyse) them open, and incubate the lysate with these beads. Then you centrifuge down the beads (with the protein and whatever's bound to it stuck to them) and wash off whatever isn't stuck to them.
To get my protein off the beads, there is also a specific amino acid sequence engineered after the "tag" that sticks to the beads. That sequence is the recognition sequence for an enzyme that then cuts the protein at that spot. So, theoretically, I add this enzyme to the beads, it does its job and cuts the protein off the beads, and I collect whatever comes off.
The problem with my project is that this enzyme is not working on my protein. I think it is because my protein is folding in such a way as to hide the specific amino acid sequence that the enzyme recognizes. I can't know for sure, because it's still pretty impossible to predict protein structure based on sequence, and I'm no x-ray crystallographer. And it's not really that important in the long run, because I should be able to get my protein off the beads by adding a low concentration of a certain chemical.
And that's the part that needs optimizing. But I'm up for it these days. Because once I have this purification scheme optimized, I should be able to generate a lot of data in a short time. The goal is to purify out my protein (+ binding partners) under different signaling conditions (i.e. after adding different proteins to my cells in culture before I burst them open), and see what comes out with it. The overarching goal is to understand how my protein regulates two different signaling pathways that have two entirely different outcomes.
Okay, I've probably lost everyone at this point. The main idea is - I'm making progress. And willing to work hard at it. I need to be out sometime during next school year. By spring of 2010, at the very latest.
Which is why I'm thinking it might not be a bad idea to start writing up the background section of my thesis, umm, now. Or whenever I happen to have a free moment. Like maybe when I'm home for 10 days over Christmas? That's why I asked P to email me a copy of his thesis. It'll be a lot easier if I have something to look at as a guide. And I can use the background section of a grant I wrote up last year as a start.
So why this renewed sense of motivation? Hmmm. Well, a girl's gotta keep some secrets, right?
And please, someone tell me if the science talk made any sense whatsoever. I'm interested to know.
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