User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/04/22/Kinesin & Microtubules

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Microtubule buffers etc.

So the buffers people typically use in bead motility assays include:

  • MgCl2
  • Some sort of pH buffer like PIPES.
  • EGTA or EDTA
  • Ha! This is called BRB80 buffer if you use 80 mM PIPES.

To fix the microtubules, they use another drug called Taxol. The next step is to coat the uncoated glass with yet another substance called Casein. I spoke a little bit about Casein and here are the major points of Casein:

  • Casein comes from milk. It is the primary source for delivering calcium phosphate to young mammals.
  • Casein forms polydisperse micelles.
  • Coating glass with Casein prevents more microtubules from sticking to the glass.
  • Casein supposedly aids in kinesin motility.

My issues with using Casein is that no one has told me why we want to use it other than it works. The problem of course is that the last paper I reviewed shows that free calcium in solution, depolymerizes microtubules. Now, they did not fix their microtubules with anything. So, the next logical question I have is,

  • Question: Does free calcium depolymerize Taxol stabilized microtubules?

I just found a paper that hopefully the library can get for me that supposedly answers this question. It has a great titleTaxol stabilization of mitotic spindle microtubules: analysis using calcium-induced depolymerization.

With that said, there is another question,

  • Question: Why does Casein aid in kinesin motility.

Well, it turns out that kinesin denatures when it adheres to glass. I just found this paper that is supposed to describe this. I found this reference from this paper (Oct. 2008).

It looks like people use Casein not for any of the reasons I thought they would. They use it so that kinesin doesn't adhere to glass and denature. I need to look into this more but at least my questions are getting more recent papers. Oh yeah, props to they guys (Vivek Verma1, William Hancock and Jeffrey Catchmark) for the open access article. You kick ass! And after the first reading of this paper, wow! You guys kick so much ass! This paper has answered a lot of my questions! You guys kick ass!.

Notes on casein

There are 4 distinct units to casein,

  • 2 [math]\alpha[/math] units
  • 1 [math]\beta[/math] unit
  • 1 [math]\kappa[/math] unit