User talk:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/09/11/Surface passivation studies
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Steve Koch 23:49, 11 September 2009 (EDT): Love the inclusions of the videos in your lab notebook. It helps a ton. OK, here are some disorganized thoughts:
- Based on what I saw with my own eyes (antifade seemed to be working; few stuck MTs; MTs that were stuck were pivoting; no directed motion of stuck MTs), I really suspect the kinesin. That is, I think of all the components in the final solution, the kinesin is bad. I'm not saying that's definitely the case, just what my gut told me. So, that would indicate (1) that the starting aliquot was bad, (2) that diluting the kinesin ruined it (did you use ice-cold buffer to dilute it?), (3) that kinesin denatured during the motility assay prep. OK, now that I wrote that out, it doesn't really close the door on anything being wrong, since kinesin is involved in most of the steps of setting up the assay. I guess what I really mean is that for some reason my gut was telling me that the kinesin in the starting tube was bad. But I wouldn't be surprised if I'm wrong either.
- I think you should try again on Monday what you did today, with a new aliquot of kinesin. I'd do this before starting over with new everything. Plus, maybe try a higher concentration of kinesin as well, just to see what happens.
- Finally, I think working together with Brigette would be a good idea. I think you could learn from each other, double-check each other, and it would be more likely to pan out. Not saying you can't make every component yourself, but you could both do it side-by-side, since you need mostly the same components.