User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/10/23/Motility

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Motility update

So I was able to get motility working again. Here's a quick run down of what's been going on.

  • Things stopped working again.
  • I got a new batch of kinesin and gave my old batch to Brigette so she can check to see if it is working or not with her bulk assay stuff.
  • I have determined that the old antifade that Brigette made back in the summer is completely useless.
  • I have determined that the new antifade Brigette made does work.
  • There seems to be a lot of attaching/detaching going on with the microtubules and the kinesin. Plus, there are a lot of squiggles in the microtubules which lends one to believe that there is a lot less active kinesin in the vials we have.
  • I have not ruled out if it is the ATP we are using as a cause for some distress. I should point out that the ATP is in PEM and not the usual Tris-HCl. I didn't see any reason why we wanted to put it in Tris-HCl other than it not hydrolyzing as quickly as it would in a buffer at a pH of 6.8. Who knows. It could be an issue. I will test this though.
  • Andy Maloney 19:29, 23 October 2009 (EDT): So I just noticed that after some time sitting on the microscope, things are moving better and more uniformly. I believe I heard Koch talk about this. Well, it turns out that the ATP is good because motility is rocking right now. I think I'm going to make sure that I don't take data for a specific amount of time. In other words, do what Koch suggested and not start until after 15 minutes has gone by after sealing the flow cell.
    • Ant did bring up a good point and that is, why has no one studied kinesin accelerating?

SJK 23:25, 23 October 2009 (EDT)

23:25, 23 October 2009 (EDT)
Hot Diggity! Glad to hear you got it working again. I do not know why (or if) people have not studied the acceleration. That one graph Larry made from your data was quite definitive and very interesting. It's also, an issue, of course if we're trying to discern slight changes in velocity. But I think it seems you know how to get motility now, and have a strong foothold (i.e. you've defeated the mysterious "stopped working" at least twice now, and I think with your care in the assays, it will actually be robust now). So, I'm really happy about that...looking forward to seeing some cool results!