User:Nadiezda Fernandez-Oropeza/Notebook/Notebook/2011/05/25

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Progress on the Motility Assay

  • This entry contains some personal remarks on the process of completing the motility assay.
  1. First try: No microtubules =(
  2. Second try: In the second trial, I got some microtubules, but really big fluorescent clusters. This might have been due to the fact that I used old aliquots of the 29% Rhodamine-labeled tubulin suspension.The following are some of the things I had to learn the hard way:
    • DO NOT leave the thermal cycler, the mercury lamp or the camera turned on!!!!!!!
    • Make aliquots of the antifade.
    • Taxol takes up to 5 minutes to defrost then you should give yourself some reasonable time before preparing the Taxol in PEM solution.
    • Ideally, the motility solution should be prepared in the 5 minutes of incubation of the Kinesin with the PEM-αA in the flow cell.
    • Try to add the PEM –Glu almost at the end.
  3. On my third trial and four trials, I got a lot of microtubules. The sample was way more populated than expected. I really don’t know why this is, I have the suspicion that it has to do with where on the tube I collected the volume of tubulin with Taxol in PEM. Discussing this with Andy, he said that it shouldn’t really matter. Furthermore, the image didn’t have a lot of contrast between the microtubules and the background. This might have to do with the way I focused the sample or maybe just with the fact that my sample was really overpopulated.
  4. On my fifth trial, the number of microtubules was more reasonable, unfortunately, I still have the contrast problem.