User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/09/11/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?
  • ATP study

Motility assay update

After many hours of failed experiments, I decided to try everything over again. I did the following:

  • Made more microtubules.
  • Got a fresh batch of kinesin.
  • Fresh stock of ATP.
  • Fresh batch of PEM-[math]\displaystyle{ \kappa }[/math]CA
  • Fresh motility solution.
  • Fresh antifade.
  • Fresh 2 M glucose in PEM.
  • Dried the beads in the e•IceBucket so that I don't have to worry about contaminating the samples in it when I touch the vials stored in it. I also decided to add some desiccant to ensure that there is no moisture build up in the bucket.
  • Polymerized for 30 minutes to see if clumping is a non issue with this batch.

Low and behold what do you know. Nothing *insert explicative* works except for the antifade.

ATP study

I've decided to not worry about the ATP study since Brian and I aliquoted ATP into 5 µL vials. This way I can have a fresh one every day of experiments.

Slide cleaning

Koch and I had a discussion about what to do with cleaning the slides for the motility assays. I decided to try 3 different slides,

  1. Dirty slide
  2. Ethanol cleaned slide
  3. Piranha cleaned slide

Dirty slide

As you can see in the below movie, I did get some motility. It is no where near the amount that I was getting 2 weeks ago.

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I didn't think about it till after uploading the video that I'm probably going to get a lot of hits with this one. Just because the name of the video is "09Sept11DirtySlide.mp4".

Ethanol cleaned slide

Well, there is no motility but there is active kinesin. You can tell that there is active kinesin by the one microtubule getting kinked.

<html> <object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/N7F6c7-wAf4&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/N7F6c7-wAf4&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object> </html>

Piranha cleaned slide

This is the Jaguar of glass cleaning. It removes everything except for defects on the glass. It is supposed to be the "bee's knees" when it comes to cleaning. As you can see, there is no motility and no motility anywhere in the slide.

<html> <object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/lGju06e3VZo&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/lGju06e3VZo&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object> </html>

Thoughts on slide cleaning

Well, I'm not sure but it looks like casein needs some sort of junk on a slide for it to stick. It could be this or it could be that the casein solution I have is bad. By the way, I am using the kappa-casein stuff I didn't make personally so I have no clue if it is bad or not. But, that is one thing that I didn't make a fresh stock of so I have no clue.

Next step

Cytoskeleton says that their ATP will last for 6 months desiccated at -20˚C. Well, it's been 5 months since we bought the stuff and we sure didn't desiccate it. So, in order to determine if it is my ATP or casein that is bad, I'm going to overload a slide to see if it is the ATP.

Andy Maloney 18:23, 11 September 2009 (EDT): Not sure it's the ATP because adding more of it didn't do anything.

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

The next thing I'm going to try is the whole casein stuff I made a few weeks ago.

Andy Maloney 18:44, 11 September 2009 (EDT): Nope. It didn't work. See the movie below. On the plus side, Brigette isn't getting here stuff to work either. The only thing we have in common is the PEM-T solution. So, it's either the PEM-T or the PEM that is causing us to have these malfunctions.

<html> <object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/6_ZbSlDT1qQ&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/6_ZbSlDT1qQ&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object> </html>

What I know right now is:

  • My kinesin is active.
  • My microtubules look great.
  • The antifade is working.

What I don't know is:

  • If the PEM is bad.
  • If the PEM-T is bad.
  • If the casein is bad.

So, the only things I can change are the PEM and PEM-T.

Working?...When?

Andy Maloney 19:45, 11 September 2009 (EDT): Koch asked me to find out when was the last time I had the gliding motility assay working. Well, it turns out it was working on 09-02-2009.

Variables

What are the variables in this experiment? I'm going to list them here.

ATP
GTP
Taxol & DMSO
Glycerin
Tubulin
Kinesin
PEM
Casein

Of these things, I'm going to list what I have made into their specific buffers.

PEM-T (recently made on 09-08-2009)
PEM-[math]\displaystyle{ \kappa }[/math]CA (made everyday)
PEM-[math]\displaystyle{ \kappa }[/math]CT (recently made on 09-08-2009)
Motility solution (recently made today)

Of these buffers I have made, all of them were made with the PEM that Linh made for us at the end of August. All of my problems started on the 3rd of September when nothing worked and all I did was update my notebook with some ideas I had. Up until the 3rd, I had enough stock from stuff made by Brigette and Linh. So, it is either the stuff I have been making or it is the PEM.

Rather than fish around to find out which thing is going awry, I'm going to make everything new. For now I think I am going to have to ensure that I do everything myself. That way if something screws up, I'm the one to blame. After I'm more comfortable, I think I will delegate.

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