User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/09/09/Surface passivation studies

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To do

To keep moving forward, I'm going to keep writing my "To do" list at the top of each notebook.

  • Hydrophobic surface.
  • Hydrophobic surface with a groove.
  • Corn starch.
  • Potato starch.
  • Gluten/flour.
  • Silica nanoparticles.
  • Silanized surface. I'm not sure how to do this one but apparently lots of people do it to attache DNA to a coverslip.
  • Lipids with PEG on a hydrophobic surface spin coated.
  • Better spin coating of lipids on coverslips.
  • PE lipids?
  • Gelatin?

One side or two?

The first thing I am going to try is to determine if I need to passivate both sides of the flow cell. I think I'm going to try spin coating casein on a slip to see what happens.

Well, we were able to spin coat a slip. The results are below.

As you can see, there is nothing on the slide. No motility was detected at all. One odd thing we did notice is that there is a rather large amount of microtubule clumping.

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After some time, I looked at the slide again and saw that some of the microtubules were stuck to the "passivated" coverslip. One thing that needs to be determined is if spin coating completely destroys the assay or not.

So I tried something. Used some tape, a sliver of a coverslip and passivated the top slip with casein. After some time, I removed the sliver and added kinesin. SJK 23:02, 9 September 2009 (EDT)

23:02, 9 September 2009 (EDT)
I didn't understand this

No motility was observed. This lends me to believe that both surfaces need to be passivated in order to have a viable motility assay. Another thing I've noticed is that if there is no casein in the microtubule motility assay, then the microtubules tend to clump together. I have no idea as to why this occurs.

Clumping

Well, I made 2 slides. 1 where I have the motility assay from yesterday with kappa casein and the other without. No kinesin was used.

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The above movie was made with kappa casein in solution. As you can see, no clumping occurred.

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The above movie was mad with no casein in solution. As you can see, clumping occurred.

I'm not very convinced that casein is needed to be in solution for the microtubules to work or not. Mainly because I made a fresh motility assay with kappa casein in it and I got clumping and no motility!

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I'm trying a new motility solution except this time I took MTs from the top of my stock solution. If there is no clumping, then I'm just going to have to make motility solutions from my stock by taking from the top of the stock.

<html> <object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/6i2uAgxWOK4&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/6i2uAgxWOK4&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object> </html>

Well, I didn't get as much clumping and there was some motility. But, there definitely is not as much as I expected to see considering the slides I made previously.

Before I can move forward, I need to understand what the hell is going on. This thing working and then mysteriously not working is really starting to irk me.

Questions about the GMA not working mysteriously

  • How long can I keep ATP out without it going bad? SJK 23:01, 9 September 2009 (EDT)
    23:01, 9 September 2009 (EDT)
    I was just writing in Ant's notebook about being careful with ATP. I know that you're supposed to be careful and that it autohydrolizes, but I don't know the details. It'd be worth knowing. Overall this is an excellent notebook page, one note, though is that if you ever refer back to it (or if an outsider is referring to it), it would be very hard to know exactly what you're doing--what kind of tubulin, how did you polymerize it, etc. Clearly we can flip back in your notebook and piece it together. But it's worth writing something down such as "same recipe as always, except this one was polymerized for 30 minutes" or "using a new aliquot of tubulin aliquoted last week." Of course, the more detail the better, but I do understand there's a limit.

    But like I said, I'm loving your open notebook. Embedded images and videos are sweet. Your commentary is great.

Update

As Koch requested, I've been working on the tubulin procedure page here. It's where all the steps I take for the above procedures in my notebook will live.

I'm too tired to work on it anymore and I think I messed some things up so I guess I'll try cleaning it up tomorrow.

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