User:Steven J. Koch/Notebook/Kochlab/2009/06/01/More FITC Microtubules, thinking about kinesin assay
See also: User:Linh N Le/Notebook/2009/06/01
Brigette, Igor, Linh, Andy, and I: Polymerized one 2 ul aliquot of FITC for 20 minutes. Stabilized with 198 ul of room temperature BRB80T. Wrapped in foil. Overall, MTs seemed less stable today, but that is just an impression.
- Could view MTs without antifade
- First attempt at antifade did not seem to do anything. Using the cocktail from last week.
- Brigette and Linh made a new cocktail: pipetting directly from the BME container (which is about 1 year old, having been stored at 4C), and using the catalase and glucose oxidase that have been freeze thawed many times since last summer. This cocktail worked much better. My impression still was that they seemed more prone to breaking up, but that's still just an impression. I think Igor commented that the MTs seemed shorter than last time.
We were contemplating trying a motility assay. However, we decided to hold off, due to one main question:
- The instructions from cytoskeleton say to resuspend with either water or a 100 mM buffer. This seemed strange to us. We're probably going to resuspend with BRB80 + 1 mM ATP, unless tech support tells us otherwise.
- Andy and I calculated that the kinesin will be about 75 micromolar if resuspended at 5 mg / ml. They were recommending 20 micromolar for the buffer, but I would prefer to have more ATP than motor domains. Thus, we're just going to go for 1 mM.
- The casein from Sigma (product# ___ ) that Andy bought was quite yellow as a powder. It did not go into solution well at all. But Andy thinks he maybe had way too much.
- The BGB I've had in a falcon tube for 3-5 years dissolved very well in BRB80 and easily was 0.2 micron filtered. We put this in the fridge.