User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/08/28/Motility assay

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Naming convention

The names of the buffers we use is pretty poorly defined. Things are labeled PEM, BRB80 etc. Since I'm going to be in charge of this experiment for the most part, I'm going to pull the "Do as I say (please)" card for a new naming convention.

I like what Cytoskeleton does with their buffers naming conventions. I'm going to spell it out here for my benefit as well as all of us who will be working on this experiment. Cytoskeleton uses the terminology "PEM" which means "PIPES, EGTA, MgCl2" in solution. We will use the same naming convention with the addition of other letters to indicate the other constituents in the buffers. So, from now on, KochLab will use this naming convention to expediate communication between all lab members. It's my hope that we will all be able to talk about the buffers with out having to have someone define what they mean in the future.

  • PEM
    • 80 mM PIPES
    • 1 mM EGTA
    • 1 mM MgCl2
  • PEM-G
    • PEM
    • 1 mM Mg-GTP
  • PEM-T
    • PEM
    • 10 [math]\displaystyle{ \mu }[/math]M Taxol
  • PEM-C
    • PEM
    • 0.5 mg/mL casein
  • PEM-[math]\displaystyle{ \kappa }[/math]C
    • PEM
    • 0.5 mg/mL [math]\displaystyle{ \kappa }[/math]-casein
  • PEM-CT
    • PEM-T
    • 0.2 mg/mL casein
  • PEM-[math]\displaystyle{ \kappa }[/math]CT
    • PEM-T
    • 0.2 mg/ML [math]\displaystyle{ \kappa }[/math]-casein
  • PEM-AT
    • PEM-T
    • 1 mM ATP
  • PEM-Gly60
    • PEM
    • 60% (v/v) glycerol
  • PEM-Gly6
    • PEM
    • 6% (v/v) glycerol

Gliding motility assay

Right now Linh is preparing more PEM for use in all our buffers. I am going to prepare a motility assay slide by;

  1. Passivating the surface with PEM-C
  2. Adding kinesin which is in PEM-CA
  3. Flowing in microtubules which are in a motility solution.

The assay worked beautifully. When we decided to try and look at the microtubules using the DIC setup, things failed miserably. Koch and I have decided to contact Olympus for help.

Andy Maloney 18:45, 28 August 2009 (EDT): I tried to compare [math]\displaystyle{ \kappa }[/math]-casein with whole casein for microtubule length. Below are the pictures.

The above picture is of whole casein.

The above picture is of [math]\displaystyle{ \kappa }[/math]-casein.

I think Hancock was right when he said that [math]\displaystyle{ \kappa }[/math]-casein supported longer microtubules.