User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/10/14/Lipid surface passivation
I am using phosphatidylcholine (PC) and phosphoethanolomine with a PEG molecule sticking out the end (PE-PEG) as a surface passivation.
All the slides were spin coated at 3 kRPM for 30 seconds with a total volume of 100 µL and at lipid concentration of 10 mg/mL. After coating, I prepared a slide and let the sample volume soak in PEM for 10 minutes followed by a rinsing of 10 µL 3 times.
Note: One thing to note is that when I went to "wash" the slides, the ones that had 100% and 99% were quite difficult in getting fluid through. Also, the other slides with 100%, 99% and 50% PC in them have bubbles. I tried really hard to not have bubbles but they ended up happening anyway.
And of course nothing is working. I can't even get motility on a regular slide. I bet it's the damn microtubules and I have to polymerize more. I don't understand. I polymerized them yesterday.
Andy Maloney 21:07, 14 October 2009 (EDT):
So after polymerizing some more tubulin, I was able to get motility working again. I should note that the slides I am using are hydrophobic. So, I should be getting many layers of lipid for the PC case. The PE-PEG, I'm pretty sure I should get a monolayer since the PEG on the head of the lipid will prevent any other lipids from making more layers. And, since I am using a hydrophobic slide, the tails of the PEG lipid should be on the slip.
So to ensure that I have everything up and running, I checked the motility assay with the standard kappa casein surface passivation.
As you can see, it is working just fine.
So the next step was to try a 100% surface passivation with PC. I was unable to get motility working but as you can see, the microtubules are sticking to the cover slip.
So what does this mean? I did see some kinks in the microtubules so I am pretty sure that some of the kinesin stuck to the lipid surface. Unfortunately, none of them were active.
I would also like to point out that the antifade just doesn't work when I am using PC. For what ever reason, the microtubules fade and even if they are stuck to the slip, they will break apart. I'm not sure if this is because the lipid surface is getting hot from the light and thus going fluid which then rips apart the microtubules before they fade. What ever the reason may be, I am very sure that using the cheap PC is just not going to work.
The next slide I did was one where I coated it with 100% PE-PEG. As I mentioned before, I am pretty sure that I have a monolayer of PE-PEG on the slip because of the PEG molecule on the lipid head.
Now, because the PEG is pointing into the solution, I am also pretty sure that there is no way a microtubule is going to get anywhere near the surface of the slide. As you can see in the movie, I think I'm justified in saying this because I just could not find a microtubule on the surface of the slips.
So, this means that using a lipid with PEG on the head is not going to work at all.
I think there are several things I do want to try before giving up on lipids. One thing is that Avanti sells lipids with a cystein head. This means that if the kinesin his-tag does interact with cystein residues on casein, then I should be able to get motility using this lipid.
I think I should also try mixtures of PC and PE-PEG as I stated before. I know that the PEG is going to prevent microtubules from coming close to the surface but the PE may help the PC from becoming fluid and thus ripping apart the microtubules.
I should also try using a regular glass slip and coating the surface with PC so I get a single bilayer. I should also try and get a monolayer of PC on one of my hydrophobic slips.
Argh! What is this magic casein protein that lets us do motility?