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

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 $$\kappa$$-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 $$\kappa$$-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.