User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/10/21/Heavy PEM

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Done

So I want to write down what has been done so that I have a list of things that still need to be done.

  • Made Heavy PEM.
  • Polymerized microtubules and fixed with Heavy PEM-T.
  • Made some Heavy PEM-κCT.

To do

  • Polymerize microtubules using the usual PEM buffers.
  • Compare antifade and ATP used yesterday to an assay that has regular PEM.
    • The reason for this is because Brigette and I were able to run one motility experiment yesterday, however, we did not see any motility.
  • If the antifade and ATP are bad, I want to replace them and try another Heavy PEM assay.
  • If nothing works with straight up Heavy PEM, then I will start to use mixtures of regular PEM and Heavy PEM in the assays.

Checks completed

So the first check needed to be made is to find out if the ATP and antifade I used yesterday is good or bad. To do this, more microtubules need to be polymerized and a gliding motility assay has to be prepared. I've decided to go ahead and use the Cytoskeleton "Perfusion Chambers" for this as well.

Unfortunately and fortunately, I saw no motility. This means one of 3 things; the antifade is bad, the ATP is bad or the kinesin is bad. I cannot believe that the kinesin is bad considering that it has only been out of the freezer for 6 days. I'm going to try again using new AF and new ATP.

After getting new AF & ATP, I tried another regular PEM motility assay.

As you can see, it still didn't work. So, on the off handed chance that the reason why nothing is working is because of the Cytoskeleton perfusion chambers, I'm trying it again with a regular old flow cell.

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