User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/08/12/Casein study
I want to make sure that everything is working, so I'm going to start off with my trusty whole casein experiment.
- Movie 1
- Seems to be not a lot of motility going on. That will change though.
- Movie 2
- I'm getting a lot of guys that have their minus ends in solution.
It really looks like my kinesin may have gone bad. I think this may be the case because I'm getting a lot of loose ends and sticking going on. Of course, it could be what I found out yesterday which was that BME is necessary for motility to work. If this is the case, then I'm screwed.
I'm going to go ahead and take an entire run just to see if the assay is robust enough to give similar speed data. I am however going to get another aliquot of kinesin and try again with a passivation that I know will work.
Since the mixed casein is basically whole casein and I know already that it will work, I'm going to try it with the new aliquot of kinesin I got. If I still see the same sort or loose ends, then I'm going to try a different motility solution with a different aliquot of antifade.
It appears that the kinesin was the issue. I have a motility assay working right now and it looks spectacular with the mixed caseins.
So here I am again with the kappa casein. I'm getting all kinds of stuck guys again. But, this time around I've decided to make movies of my ROIs. This way I can tell if the MTs are stuck completely or if they are just stuck sort of and will eventually move.
So for each region, there is on average zero motile microtubules. However, there are some...sometimes. I should try a 0.5mg/mL passivation and a 0.25mg/mL in the kinesin assay for fun.
I need to point out that at movie 18, I moved the FOV to close to the tape. I have motility. I'm going to set aside this slide till after I finish with my beta casein prep.
So for this assay I used the cold block of aluminum for incubation. I'm not sure if it is the kinesin or the cold block but, I do get motility right off the bat. And, it is in the center of the flow cell. I should point out however, that there are not that many MTs in the center of the flow cell.
I am getting motility. It's sparse and I have to hunt for decent areas to take data in.