User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/11/03/Microtubule studies

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What to measure

So I'm a bit confused about what measure I should use to determine the differences between Heavy TSB polymerized microtubules and Regular TSB ones for stability purposes.

  • I could do absorption measurements with the NanoDrop over time.
  • I could do photobleaching experiments.
    • How to normalize this is tricky.

Stability

So both the regular and heavy stuff are still around. I think it has a lot to do with using fresh Taxol every time you go to polymerize the guys and to make a motility solution.

I decided to see if I could destroy some microtubules with a bunch of light. Unfortunately, I couldn't do it. I have no clue why but they just wouldn't break. At any rate, what ended up happening was fading which is a good fluorescence study. The specifics of the below movies are,

  • Playback rate = 30 Hz
  • Total time = 15 minutes
  • Exposure per frame = 100 ms
  • Gain = 150
  • Full Hg lamp power.

What I find amazing is that the stuff in Heavy motility and polymerized in Heavy TSB is crazy bright. That's why it is a white screen for so long. This of course means that if you were to use less power, you would be able to keep these guys around for much longer than the regular motility solution. Plus, even after 15 minutes of exposure, you are still able to discern the microtubules in the Heavy stuff. Not so with the regular sauce.

I should note that sometimes the images look like they are getting brighter. This is because the focus of the microscope changes due to thermal gradients.

Regular sauce

<html> <object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/bpskG6mGAYs&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/bpskG6mGAYs&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object> </html>

Heavy sauce

<html> <object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/SQWl7pv6p44&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/SQWl7pv6p44&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object> </html>

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