Wow, last week was memorable. Not only did I sign my contract with the University of Toronto, but it appears as if my super duper theory that I thought I had killed, might have been resuscitated.
To remind you, the last time I wrote about my trials and tribulations, I thought that I had ruled out my super duper theory because of a simple straight forward experiment. The experiment involved microinjecting a dominant negative protein that blocks a complex from working. What I failed to tell you is that I was waiting for my positive control, a protein whose distribution would be altered if that magic complex was indeed blocked. Finally I got my hands on the positive control, however the injection of the dominant negative protein did not disrupt the distribution of my dominant negative protein.
(my artistic rendering of a cell and of the central dogma covered with several subsequent scriles and a few calculations. The two surrounding taped messages are from baymate.)
I thought to myself what's going on here?
So I then went back and reread the literature.
When doubt, go back to the original sources (and their materials and methods).
It turns out that previously, folks had performed this experiment slightly differently. I thought to myself, that shouldn't make much of a difference, in fact, the way I performed the experiment should lead to a much more dramatic result. But this is biology, so who knows.
Thus I attenpted to repeat the experiment in the exact manner that it was done before, the only difference is that I used a different cell line. The result? Thankfully, the dominant negative protein injection worked as the distribution of my "positive control" was altered, however I have to say that the effect was very weak. Hmm, some cell line differences perhaps? That's very interesting on several levels. But at least I know that the dominant negative protein really acts AS A DOMINANT NEGATIVE.
So what about my super duper experiment?
I redid that as well, notr expecting much. However to my surprise, it worked beautifully, my process was blocked completely. 100%. But before I begin my victory dance, I'll want to perform a few negative controls. I'll need to show that the cells are not completely screwed up by demonstrating that other related processes are working just fine.
I'll let you know later this week.
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First thing I thought of when I saw your picture was this http://www.venganza.org/him2.jpg :)
Yeah ... I tend not to trust the efficacy of dom neg constructs. When they "work" they work; when they don't work it might be due to any number of things, you know?
Good luck, though.
Not to mention, if you're testing a signaling molecule like a GTPase or a system which relies on a kinase/phosphatase cycle, the dn might have the same phenotype as the ca, or an unexpected phenotype, because you're jamming the works. Hang in there. It wouldn't be biology if it weren't more complicated that we thought it was! You'll figure it out.