User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/10/15/Lipid passivation
After a restless night thinking about this stuff, I have come to the conclusion that I do not want to use lipids that have PEG on them. Mainly because I know that there is no way a microtubule will ever reach the surface of a slip due to the PEG.
Also, I realize that the PC I am using is really not all that great. After emailing Avanti, I was able to get a link to a table that shows the melting temperatures for most of their lipid catalog. I really think that the light from the mercury lamp heats up the lipids enough to make them transition from their gel state, to their fluid state. This is a problem because I want a rigid surface that will not break apart the microtubules if they are on the surface. Also, I don't want the kinesin sloshing around the lipid membrane causing funkiness in the motility assay.
So, after looking at Avanti's table, I chose to get some 18:0 PE which has a melting temperature of 74˚C. I also got some cationic 18:0 TAP. The reason for getting this is because I'm still interested in why kinesin will attach itself to casein. Of course the physicist in me wants to look at the simplest possible reason and the idea I came up with is charge. We shall see if I can get any insights from using these lipids.
Since I have PC and PE-PEG, I am going to run a few intermediate points of lipid ratios. From yesterday, I should expect that there will be next to no microtubules sticking to the slide in the 1:3 PC:PE-PEG, some in the 1:1 PC:PE-PEG and more in the 3:1 PC:PE-PEG.
Again, all slides were spin coated at 3 kRPM for 30 seconds with a total of 10 mg/mL lipid concentration. I used 100 µL of the 10 mg/mL lipid solutions to coat the slide completely.
I did realize I made a mistake only now. Thankfully the below experiments I fixed the mistake, however, the ones I did yesterday had this mistake. I made a motility solution with PEM-kCT when I should have made it with PEM-T and no casein. Today, I made some up with my polymerized microtubules from yesterday.
Before I start doing anything with the slides that I prepare, I always check the motility is working before using up the spin coated slides that take hours to prepare. This was an unfortunate lesson learned. At any rate, it seems that the microtubules that I polymerized yesterday are no longer good. I have no clue as to this reason seeing that they usually last quite a long time.
The MTs not being viable could be because I took from the top of my stock solution. I will prepare another slide to see if this is the reason.
MT stock check
Nope. They are totally toast. I have no clue as to why this is occurring but I have to polymerize more now. I'm guessing my stock solutions of with Taxol in them must be contaminated somehow so I'm going to make more.
Andy Maloney 18:38, 15 October 2009 (EDT):
After making more PEM-T & PEM-CT, I was able to run a motility assay. Unfortunately I do not have enough active kinesin so I'm getting another tube of it from the freezer. I guess I will try again.
Andy Maloney 19:11, 15 October 2009 (EDT): And again.
So this time it worked. One thing I should point out is that I thought that the motility assay was not working. Mainly because I use the 1.6x magnification thingy on the microscope most of the time. This time I didn't and I thought that the microtubules were moving too slowly. Well, after engaging the 1.6x thingy, they moved as I remember them doing. So, another thing I should have known that I know now.
Now more lipid stuff
So I want to make sure I write this down so that I can reproduce it with other slides. I am going to do it like this from now on.
I wash the slides with PEM twice and let it sit for 10 minutes. I then wash it again twice before adding kinesin. After 5 minutes, I add the motility solution that does not have any casein in it.
25% PC & 75% PE-PEG
So I saw no motility with this slide. I did see a lot of depolymerization going on. I think that the plastic slides do something funky to the assay. Or, it could just be getting too hot. Since there was a lot of PE-PEG on the slide, I didn't see any microtubules on the slide surface.
50% PC & 50% PE-PEG
Again, I did not see any motility. I am wondering however, if the kinesin did stick to the PEG and the microtubules were sticking to the kinesin. But, since the mercury lamp gets the sample hot, the PEG starts to move around more and thus toasts the microtubules.
I will have to think of a way of detecting this.
75% PC & 25% PE-PEG
Again, no dice.