User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/11/25/Sucrose
- I polymerized microtubules.
- I put 2 M sucrose in PEM.
- I prepared from the 2 M sucrose solution, 3 dilutions of 0.25 M, 0.50 M, and 0.75 M sucrose in the motility solutions.
- I made my flow cells.
So I did change some things up from my heavy water experiments. Instead of doing the sucrose dilutions sequentially in time, I prepared all of them at the same time. I did however, not flow in the microtubules until after I took data for each slide. This may have caused me some issues with the 750 mM sucrose guy. I'll have to redo this to make sure. Suffice it to say though, the 250 mM sucrose guy does look like the MTs are moving slower and that's all I was hoping to see today. I will have to redo it anyway in a cleaner fashion to make sure I didn't do something silly.
All movies are 2 minutes long for one field of view (I took 3 FOVs). The playback rate is 5 frames/s and the capture rate was 5 Hz so the movies are not sped up. The images have an exposure of 100 ms and a gain of 150.
250 mM Sucrose
500 mM Sucrose
750 mM Sucrose
These guys definitely look like they are running slower than the motility assays I have done without osmolytes in them. Of course, I cannot determine how much slower until I analyze the movies made.
- Steve Koch 23:41, 25 November 2009 (EST): The first two are definitely slower than normal. Did you do a 0 mM control? That would be necessary to make sure something else funky isn't going on. The 750 mM doesn't look like it's moving at all. And it seems to be a repeatable time to synchronous opticution. Caused by sucrose? Maybe so: it could inhibit the antifade. That's something I definitely haven't been thinking about enough. The antifade is a dual-enzyme system and will be affected by all the things we're trying to do to the kinesin with osmotic stress or heavy water. That even more complicates the interpretation of "long-lived" and other effects. Hmmm. We should look into the uniblitz shutter option (for time lapse with much less illumination).