The last week of this rotation contained many ups and downs. On the positive side, I produced the most beautiful western blot I've ever seen. My tricky protein finally behaved, and I was able to detect nice, sharp bands in most of the samples I tested. I was so excited! I showed the postdoc who's been helping me, and she was also excited. I showed my adviser, and she was pleased. Yesss! I am a western blot queen! All I had to do was repeat the experiment two more times, and I've have some quantifiable data.
Well, you know what they say about the best-laid plans of mice and scientists... I ran the gels again and the new blots looked like crap. I wasn't able to get any signal from the samples that had behaved so beautifully the previous day. I'm told that this is a common problem that people in the lab have with this protein. We hypothesize that perhaps the protein doesn't like freeze/thaw cycles, and thus it only shows up when freshly made protein lysates are used. That's pretty annoying, since I get about one mL of protein lysate from each mouse tissue, but only use 10-50 µL for each gel (thus, each tube of protein lysate should be enough for 20-100 experiments!). One potentially easy way to solve this problem, if it is a freeze/thaw thing, would be to aliquot my fresh protein lysates into 20 individual tubes, instead of one big tube, so that I'd only thaw what I need for one experiment at a time. Doing this is somewhat annoying, but if it preserves my ability to get beautiful western blots, it's worth the trouble. However, with my rotation now over, I don't have time to spend three days making another set of lysates, aliquot them, and try again. Instead, I will be creating a rotation report based on very little actual data. My methods section will be long, though -- I tried a lot of different things to make this protein cooperate with my experiments. A few of them kind of worked. I can document that.
I start my third (and probably final) rotation on March 16. No more rotation diaries until I start working there.
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