User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/03/04/MT speed and passivation

New solutions
I had to remake the following solutions:
 * Antifade
 * PEM
 * Taxol
 * Microtubules

Experimental outline
The experiment will consist of: I will look specifically for any speed variations and also the type of microtubules that are exhibiting motility in all the different assays. If I have time, I will passivate surfaces with BSA, PVP, and PEG to see what happens as well.
 * Motility with whole casein passivation.
 * Andy Maloney 12:30, 8 March 2010 (EST): Assay finished. Data analysis to come.
 * Motility with alpha casein passivation.
 * Andy Maloney 12:30, 8 March 2010 (EST): Assay finished. Data analysis to come.
 * Motility with beta casein passivation.
 * Andy Maloney 12:30, 8 March 2010 (EST): Assay finished. Data analysis to come.
 * Motility with kappa casein passivation.
 * Andy Maloney 12:30, 8 March 2010 (EST): Assay finished. Data analysis to come.

Note

I should note that I have not been setting up proper illumination with my Hg lamp till now. This is a big oversight on my part but it should not affect the data I have take previously. I just don't get the best images possible.

Andy Maloney 12:30, 8 March 2010 (EST): I have now started setting up Kohler illumination from my Hg lamp. I have also started making the aperture from the Hg lamp just slightly larger than the FOV in the camera. This will help prevent photobleaching of the MT not in the camera's FOV.

Experimental details
The details of the experiment will be:
 * Use 25% Hg illumination from my 100W lamp.
 * I will take 15 ROI in each slide prepared and I will record the times accordingly so as to take data from each prep at the same approximate time the slide has been illuminated... explain better
 * Each ROI will take 600 frames at 5 frames a second for a total approximate time of 2 minutes in each ROI.

Experimental results
I am hoping to post characteristic images of each slide prep as well as a characteristic movie. Data analysis will have to come later.

Whole casein
Andy Maloney 16:16, 10 March 2010 (EST): Below is a characteristic image of my whole casein prep. The image is false colored with ImageJ. As you can see, there are really small microtubules in the image. They actually exhibit motility.



One interpretation to this would be that there is a high concentration of kinesin on the glass. Since such small microtubules can crowd surf, this means that whole casein can somehow pack kinesin dense enough to allow this small microtubule to move. Below is a movie from a different region than the above picture. This area still has really small microtubules moving along.

   

Alpha casein
Andy Maloney 16:26, 10 March 2010 (EST): Below is a characteristic image from a slide passivated with alpha casein. This to can support small microtubules.



This is a movie showing that the microtubules exhibit motility using alpha casein. It is not from the same region as the above picture.

   

You can see that in the movie I do get a some really small microtubules exhibiting motility. To me, this means that alpha casein is somehow packing the kinesin tightly enough to support such small microtubules.

Beta casein
Andy Maloney 16:37, 10 March 2010 (EST): Below is a typical image with beta casein. I will note that when using beta casein, the microtubule minus ends tend to come off of the glass a bit more than when using whole or alpha casein. I'm not sure why this would happen but you can see it in the below picture.



This movie is not from the same region as the above picture.

   

You can easily see that some microtubule minus ends come off of the glass. This, I really have no clue as to why this happens. Now what's really cool is the video below.

<param name="movie" value="http://www.youtube.com/v/b73JZo6myi4&hl=en_US&fs=1&rel=0"> <param name="allowFullScreen" value="true"> <param name="allowscriptaccess" value="always"> <embed src="http://www.youtube.com/v/b73JZo6myi4&hl=en_US&fs=1&rel=0" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="480" height="385">

In this video, you can see a few small microtubules exhibit motility. But, the important thing is that there is not very many of them and they inevitably come off of the slide before then end of 2 minutes. This seems to mean that there is not enough kinesin on the slide to support motility for the smaller microtubules.

Kappa casein
Andy Maloney 16:58, 10 March 2010 (EST): Now here's the kicker. This is a slide passivated with kappa casein. As you can see, there are no small microtubules around.



As you can see in the below movie, the surface cannot support small microtubules. Plus, I finally understand what Steve meant when he thought that the kappa casein slides showed "sticky" microtubules.

<object width="480" height="385"><param name="movie" value="http://www.youtube.com/v/E9EWryqDgNY&hl=en_US&fs=1&rel=0"> <param name="allowFullScreen" value="true"> <param name="allowscriptaccess" value="always"> <embed src="http://www.youtube.com/v/E9EWryqDgNY&hl=en_US&fs=1&rel=0" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="480" height="385">

Speeds
Below is a Google Doc showing the speeds for the tracked microtubules from the above data.

<iframe width='800' height='300' frameborder='0' src='http://spreadsheets.google.com/pub?key=tJAEUMW-ZPXCzzJcahyY6hw&output=html&widget=true'>