User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/08/10/Casein study

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Kappa casein 1st try

So I was able to see motility for a very small number of MTs in this assay. Not sure, but it could be that my ATP is bad even though I got a new aliquot from the freezer. To make sure it is okay, I'm going to try a whole casein assay.

Why doesn't it work?

I'm not sure why this passivation doesn't work. I think that one reason may be due to what kappa casein is. It is a glycoprotein and it has very different properties than the other caseins. People think that this glycoprotein is on the shell of a casein micelle and it allows the micelle to be in solution due to its amphiphilic nature.

There are two things that I think may be going on.

  1. Kappa casein does protect kinesin from the glass surface but, think of kappa casein as tall grass. The kinesin may have its motor domains free to grab a microtubule but, the casein is preventing the neck linker from going through a conformational change thus not allowing a power stroke. This makes some sense due to the fact that I get a ton of stuck microtubules and I have some that are motile.
  2. Kappa casein does not support the kinesin so that its motor domains remain active. Kappa casein actively binds to the kinesin in a way that prevents motility.

I'm not sure which one of these makes more sense. Nor am I able to distinguish the differences in my setup. What I do know is that I have observed the following:

  1. I get a lot of stuck microtubules when using the kappa casein passivation. Those stuck microtubules look squiggly.

Whole casein 1st try

I am coming to the realization that beta and kappa casein just don't work consistently enough to be reliable. At any rate, I still need to do 2 more whole casein experiments and this is counting as one of them.

The ATP and motility solution are the same for this experiment. The only difference is that I'm using whole casein as the surface passivator. I should also note that all experiments are being incubated at room temperature as I'm not convinced that the hot plate/cold plate are doing much of anything.

What I think is going on is that kappa casein is hindering kinesin from being motile. Also, I think the "stickiness" that we see sometimes in the whole casein assay is due to the kappa casein. I need to explain this more and think about it more.

So I was able to start the whole casein passivation off with no problems at all. This means my ATP is just fine.

Beta casein 1st try

So this time around I didn't cool or heat the slide before going on the microscope. I'm getting motility that is characteristic of the beta casein assay. Long microtubules that tend to have their minus end in solution.

Also, the closer one gets to the edges of the tape, the more MTs there are moving around. This is a catch 22 situation. I know from previous studies that the closer I get to the edges of the flow cell, the slower my microtubules will glide at. But, it looks like the only spots in the flow cell that are supporting motility are those that are near the tape edges. This is why using beta casein is not a good idea. While it may be the best in terms of purity, one will never get consistent data if you take it near the tape edges.

Whole casein final assay

So this is my final assay using whole casein and temperature stabilization.

  • Movie 9
    • Ha! There is one with a gigantic tail.

Alpha casein final assay

So again, this is the final assay using alpha casein for this experiment. I seemed to have a few issues for the first 2 movies but now I'm not. It really looks great.

  • Movie 12
    • There is a guy with a huge Taxol crystal on its minus end.
Steve Koch 20:51, 10 August 2010 (EDT): The taxol issue (and that good PLoS ONE paper from last year describing the solubility issue) is one to keep in mind. If the affinity of taxol for MTs is very high (and it might be), then you could potentially greatly reduce the concentration in storage and motility buffer. You would stabilize the MTs with warm PEM-80T, but not dilute very much. Then, after stabilization, you would dilute into storage buffer with less taxol. There may be molarity and other issues to consider. And it's not urgent to do this, just something to keep in mind.
Andy Maloney 12:44, 11 August 2010 (EDT): It's actually not a problem, I was just noting it. I have my assays setup is such a way that the Taxol crystal problems don't occur.