User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/08/12/Surface passivation trial 2

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Trial 2

So nothing seemed to work yesterday. I plan on looking at something that I know will work and then I can determine if the new stuff I am going to do will have the chance of working.

I have no idea why I didn't do this first yesterday. From what everyone is telling me, kinesin and microtubules are labile and can screw up pretty easily. Why I didn't make sure everything was working before I tried something new is beyond me.

Plan

  • Make a κ-casein coated slide and make sure everything is working.
  • Prepare a lipid slide using what the paper says to use, 1 mg/mL lipid concentration.

First step

The first thing I want to do is make sure I still have microtubules. To do this, I prepared a "motility solution" since I know I will be using kinesin today. The hope is that I can take an image of the microtubules to ensure that I have some to work with today.

I also got a batch of fresh anti-fade for use in the motility solution.

As you can see with the above image, I do have microtubules and they are stuck to the glass.

Second step

The next step is to see if I have active kinesin. To do this I will;

  1. Prepare a slide coated with κ-casein.
  2. Flow kinesin in.
  3. Flow MTs in.
  4. Observe.

This worked out beautifully. I will post an animated gif once I can do this.

Third step

  1. Coat a cover slip with 1 mg/mL lipid molecules.
  2. Hydrate with BRB80 in a flow cell.
  3. Add kinesin.
  4. Add MTs.

Well, I forgot to add the anti-fade. Thus, the slide is not viable. It does look like there are globular formations on the slide and I believe this is from the lipids aggregating. One thing to note is that the paper I read indicated that the slides they prepared were under vacuum for 20 hours. I'm not sure this is entirely necessary but it may be the case.

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