User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/09/02/Surface passivation studies
Again, we spin coated 100 µL of 1 mg/mL phosphatidylcholine onto a glass cover slip. We spin coated the slip at 3000 rpm.
We also made sure everything was working by redoing a gliding motility assay. Below is a movie of the assay which has a kappa-casein coated surface.
I should note that we tried to spin coat the lipid molecules on half a cover slip by protecting the other half with a piece of tape. I then removed the tape and prepared the slide as usual. Also, since we spin coat the slip, the passivated surface is not on the slide. I'm not sure if this changes the imaging or not so I will have to try one out with the kappa-casein protecting the slip and not a slide next.
I unfortunately uploaded these videos to my own account and not to the KochLab channel. I'll have to change that some time later. As you can see, the microtubules are not moving around. In fact, they are stuck to the lipid areas which I think are the light gray areas. What is interesting is that this assay causes a catastrophe to occur where the microtubule basically disintegrates. This definitely doesn't bode well.
I'm going to try again any way and see if I can't make a slide more carefully.
kappa-casein on the slip
I went ahead and made a kappa-casein coated cover slip to see if I was causing optical artifacts when I spin coat lipid molecules onto a slip and visualize them. Nope.
It definitely looks like I have lipid garbage on my cover slips and that garbage is the light gray stuff in the second movie where a microtubule is destroyed.
I was thinking about this problem last night and I think the reason we have the weird splotches on one side is because of the tape. We are either getting some under-spillage, or pulling up the tape is causing a wicking effect. Also maybe the tape is leaving some glue residue on one side as well. Lets try and spin coat on the cover slide, but let's make two sample areas. Basically have a spin-coated coverslip and make a channel out of that, and have another non spin coated coverslip with channels on another part of the slide. Let's mess around with this later.
Andy Maloney 00:54, 3 September 2009 (EDT): I'm all for trying this tomorrow. I also want to have a slide with nothing but lipids on it to see if that works or not. Also, I think we will need to coat both surfaces (slip and slide) with the lipid molecules. Not sure though.
Where does the passivation occur?
Well, I asked myself this question. The reason I asked myself this is because I was spin coating lipid molecules onto a glass cover slip. That slip was then placed on a glass slide (no passivation) with 2 pieces of double stick tape to create a flow cell. I thought that I had to keep the slip and slide inverted to ensure that when I flowed in kinesin, the kinesin would "fall" onto the lipid surface and thus I would be able to make a gliding motility assay on that surface. This procedure is exactly opposite to what we do normally. A picture is worth a thousand words... This is an image of my lipid flow cell. Everything is upside down relative to the casein flow cell.
I thought that I needed to have everything upside down because of my belief that the kinesin only attached itself to the lower glass surface. In the case of the lipid coatings, it would be the slip but in the case of the casein coating, it was the slide.
It turns out that I'm wrong and that both surfaces get coated with casein when you flow it into the flow cell. Kinesin also attaches itself to both the slip and the slide as can be seen with the following movies.
The above movie shows that the gliding motility assay works when focused on the slide surface. The below movie is of the same sample except it is focused on the slip surface.
As you can see, both glass surfaces support the gliding motility assay. This raises an important question for the lipid surfaces: Does both surfaces need to passivated for a gliding motility assay to work?
I do want to point out several cool things about the last two movies above.
- In the last movie you can see a big microtubule and a small one moving parallel to each other. Note that they are moving at the same rate. I know we already know that they should move at the same velocity but, it is kind of cool to see it.
- In the second to last movie you can see the microtubules coming off of the substrate and wiggling around a bit. I think this is because of a kinesin motor losing its grip on the microtubule and that end of the microtubule going into solution (i.e. away from the glass substrate).
- We could probably measure some quantities about our microtubules from the second to last movie because you can see on get a kink in it without breaking.