User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/10/13/Lipid preps
I have 2 types of lipids here. One is phosphatidylcholine (PC) and the other is phosphoethanolomine with PEG attached to it (PE-PEG). I am going to use both of them for surface passivation studies.
The first thing that I need to do is exchange the chloroform that the lipids come packed in with a 1:1 solution of ethanol and methanol. The reason behind this is that the chloroform will eat the hydrophobic slides that I have and in order to spin coat the lipids onto the surface, they must be in solution. So, I am exchanging the chloroform with 1:1 MeOH:EtOH which does not eat the slides.
Exchanging the chloroform is really easy. You just blow away the chloroform and reconstitute the lipid molecules with something else. Of course, doing this would be best suited for using a Roto-Vap but I'm using the one I made.
For the experiment to see if motility works, I am going to try these things:
- Make sure my stuff is still active by reproducing the casein experiments.
- Spin coat 100% PC onto the slides.
- Spin coat 100% PE-PEG on the slides.
- Spin coat 1% PC and 99% PE-PEG.
- Spin coat 99% PC and 1% PE-PEG.
- Spin coat 50% PC and 50# PE-PEG.
I think I read somewhere that PE has a melting temperature above room temperature. If this is the case, then the PE-PEG will produce a somewhat solid layer on the slides. Of course, the PEG will make the interface between solution and the lipids a "fuzzy" interface so I'm not entirely sure that any microtubules will come near it even if I get kinesin to stick to the lipid/PEG surface. We shall see though...
Well, after several hours of prep, Brian and I were able to see that the motility assays are still working.
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Unfortunately, I have run out of time for right now. I have stored the prepared slides in the refrigerator in a bag filled with desiccant. The reason for this is because I do not want the lipid surfaces coming anywhere near a lot of water. I want to do this to prevent them from doing anything funky before I am able to add the motility solution.
Well, I'm back and I guess I'm back to looking at the lipid stuff. I should note that I am going to flush the chambers out with PEM before introducing the kinesin. I think a good soak of 10 minutes and a wash will be sufficient.
One thing to note about the below is that the antifade has gone south. This is a good lesson learned. I made a motility solution and let it sit for about 3 hours before I was able to use it. If you make a motility solution, then you had better use it within 1 hour. Otherwise, the antifade will go bad and you will either have to add more antifade or make a new batch of motility solution.