User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/01/26/Caseins

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Solutions

I have now solutions of:

  • 0.5 mg/mL alpha-casein in PEM (α-PEM)
  • 0.5 mg/mL beta-casein in PEM (β-PEM)
  • 0.5 mg/mL kappa-casein in PEM (κ-PEM)
  • 0.5 mg/mL whole-casein in PEM (W-PEM)
  • 25 mM sialic acid in PEM (S-PEM)

New stock solutions

I had to get some new stock solutions and they are:

I also need to polymerize new microtubules and I will have to wait till the thermal cycler is available.

Experiments

These are the following experiments that I want to accomplish today and possibly tomorrow if I run out of time today.

α-PEM

I need to make:

  • PEM-αT
  • PEM-αA

The experiment will consist of:

  1. Passivation with α-PEM.
  2. Store kinesin in PEM-αA
  3. Make a motility solution with PEM-αT.
  4. Determine if motility works with α-PEM
    1. Determine the speed change (if any) using alpha-casein.

α-PEM Results

<html> <object width="445" height="364"><param name="movie" value="http://www.youtube.com/v/Zgg1NXrJMek&hl=en_US&fs=1&rel=0&border=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/Zgg1NXrJMek&hl=en_US&fs=1&rel=0&border=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="445" height="364"></embed></object> </html>

  • Update Andy Maloney 11:21, 27 January 2010 (EST): Today I will post a typical moving showing motility. I do want to note that in this assay, the antifade didn't work all that well.

β-PEM

I need to make:

  • PEM-βT
  • PEM-βA

The experiment will consist of:

  1. Passivation with β-PEM.
  2. Store kinesin in PEM-βA
  3. Make a motility solution with PEM-βT.
  4. Determine if motility works with β-PEM
    1. Determine the speed change (if any) using beta-casein.

β-PEM Results

<html> <object width="445" height="364"><param name="movie" value="http://www.youtube.com/v/Ofxk7U7g71Y&hl=en_US&fs=1&rel=0&border=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/Ofxk7U7g71Y&hl=en_US&fs=1&rel=0&border=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="445" height="364"></embed></object> </html>

κ-PEM

I need to make:

  • PEM-κT
  • PEM-κA

The experiment will consist of:

  1. Passivation with κ-PEM.
  2. Store kinesin in PEM-κA
  3. Make a motility solution with PEM-κT.
  4. Determine if motility works with κ-PEM
    1. Determine the speed change (if any) using kappa-casein.

κ-PEM Results

Andy Maloney 14:46, 28 January 2010 (EST): After much fuss. I was able to determine that my kappa casein has gone bad. I will have to return to this at a later date when I have gotten some more kappa casein.

W-PEM

I need to make:

  • PEM-WT
  • PEM-WA

The experiment will consist of:

  1. Passivation with W-PEM.
  2. Store kinesin in PEM-WA
  3. Make a motility solution with PEM-WT.
  4. Determine if motility works with W-PEM
    1. Determine the speed change (if any) using whole-casein.

W-PEM Results

<html> <object width="445" height="364"><param name="movie" value="http://www.youtube.com/v/DNSu-LhiQ6U&hl=en_US&fs=1&rel=0&border=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/DNSu-LhiQ6U&hl=en_US&fs=1&rel=0&border=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="445" height="364"></embed></object> </html>

S-PEM

I need to make:

  • PEM-ST (1 mM sialic acid)
  • PEM-SA (1 mM sialic acid)

The experiment will consist of:

  1. Passivation with S-PEM.
  2. Store kinesin in PEM-SA
  3. Make a motility solution with PEM-ST.
  4. Determine if motility works with S-PEM
    1. Determine the speed change (if any) using sialic acid.

S-PEM Results

<html> <object width="445" height="364"><param name="movie" value="http://www.youtube.com/v/xzLCk_iuqqg&hl=en_US&fs=1&rel=0&border=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/xzLCk_iuqqg&hl=en_US&fs=1&rel=0&border=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="445" height="364"></embed></object> </html>

  • So no motility. However, I have seen a few MTs depolymerize but when they are gone, there is a ghost image of where they were.
  • Another odd thing is that there seems to be ghosting effects on some MTs but only on one end. Never on both.
  • There also seems to be a rather large number of MTs sticking on their ends. I see dots on the microscope slide and then when I go farther into the sample, I see a diffusing dot (end of movie).
  • Note: So not getting motility using just sialic acid is actually not a bad result. It does tell me that there is stronger evidence that motility is aided by some sort of structural component to the caseins. I do have an enzyme that can cleave all the sialic acid residues on all my different caseins. If I use this, and find out that I can still get motility, then the sialic acid residues are not what maintain motility. Although, it seems that they do attract the ends of microtubules.

Thursday Update

Andy Maloney 14:03, 28 January 2010 (EST): So on Wednesday, I tried to complete this experiment and unfortunately got hung up with my antifade not working. So, I made some more and tried again with my kappa-casein.

Unfortunately, it took some time but I have figured out that my kappa casein has gone bad. I will need to reorder some but thankfully, I can continue with the sialic acid portion without having to take the kappa casein data right now.

Steve Koch 22:08, 28 January 2010 (EST):Some of these movies look just spectacular! -- especially the whole-casein just looked like very nice motility. You're really good at these assays. To put down some of my notes from (Wednesday?):
  • We looked at several of your movies (alpha and whole casein I believe), and we found some results:
    • Looked like speed right after preparing the assay was slower than after a while. I think we agreed that like others, we should use later video for the "final" speeds. But you should still take the early videos, because maybe someday we'll understand why there would be this strange acceleration. Particularly if you follow one MT for a long time, like you've done in the past and can see if individual MTs accelerate.
    • We saw evidence of speed associated with microtubule identity. Our best hypothesis, which I still like, is that the intrinsic speed has to do with the protofilament number. You looked this up and under our polymerization conditions, we'd expect >90% of the MTs to be of two types. In fact, we did seem to see two populations of speeds. Larry has now improved the tracking software so it's quite a bit faster. I think you have enough data from these sets of experiments to really look into that, and it could be quite fascinating.
  • As for software, like I said, Larry made many improvements and it's now much faster. We're still a bit unsure how to store the data, but I think from working with you we are more clear. We had a good idea today to save individual results to a spreadsheet file -- maybe even automatically to a google doc, which would be quite slick, huh?
    • Andy Maloney 01:41, 29 January 2010 (EST): Yes it would be. Or maybe to a text file R can read. Looks like I will be learning R.
      • Steve Koch 12:34, 29 January 2010 (EST): Learning R is a good idea for your career I think. Richard likes it and uses it in his job. Lots of people on friendfeed use it. The file will be easy to save as both Excel and tab-delimited text file, so that's easy.
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