User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/08/07/Passivation study
So I'm going ahead with the belief that keeping the slide cold before putting it on the objective is the way to go. I've also noticed that there is not as much evaporation of material if the slide is on the cooler. Of course this is obvious but I didn't think about it previously.
Whole casein 1st try
- 1.0mg/mL whole casein in PEM for 10 minutes on the cold plate.
- 0.5mg/mL whole casein in PEM + 1mM ATP + 27ug/mL kinesin for 5 minutes on the cold plate.
- Motility solution.
I should point out that it took me longer to get to the first ROI due to improper placement of the slide on the microscope. But, everything seems to be working just fine right now.
- Movie 2
- So far there is a mixture of small, medium, and long MTs exhibiting motility. I will note that the focus is drifting a little bit. This means that there is still some temperature gradients going on.
- Between movies 5 & 6, I had to readjust the slide so that the images were in focus for the entire FOV.
I'm getting some squiggles and some circles reminiscent of the heavy water studies.
Alpha casein 1st try
I really think it is the cold plate that allows me to get passivation. I was able to see motility with the alpha passivation with relative ease. As opposed to when I used the hot plate.
I am having zero problems with this new setup.
- Movie 3
- There is a rather odd MT in that the the plus end of it was attached to the kinesin surface but the minus end was in solution. Very odd looking.
- The focus isn't drifting that much with the alpha casein.
Beta casein 1st try
Moving forward to the next assay.
It seems that the only spots where I'm getting motility are near the tape in the flow cells. This is not a new characteristic when using beta casein.
After looking at all the assays before where I used beta casein, none of them look very good. I think that the data I did get before was for MTs that were near the tape. As I already know, gathering speed data near the tape of the flow cell is not the best thing to do. It appears that beta casein is not a good choice when running these assays. Some things I found include
- Most all of the MTs are super long.
- There is typically not very many in the assays.
- I get a large number of stuck ones that fishtail.
- There are also a large number of them that have their minus end in solution while moving.
I believe the past 3 weeks have convinced me that beta casein, while it works, is not very good at passivating the glass surface. So far, alpha is winning in terms of ease of use and the gliding motility assay.
Kappa casein 1st try
So I already know that in previous experiments, the kappa casein assay doesn't work very well. I'm going to see if it behaves as expected here.
Yes. As expected, the kappa casein passivation is not working at all.
So this is turning out to be interesting. I started this project thinking that all the caseins would support motility. It turns out that I'm only able to get it to work with 2 caseins in an efficient and reproducible manner, the whole casein and alpha casein.
It seems that the only spots where I'm getting motility is near the tape with this casein. Very similar to the beta casein study. Although, there are differences.
Thoughts on the caseins
This is very interesting. Whole casein is the toughest to get into solution. The next most difficult one is alpha casein. I can't recall so I'll have to look it up but I think I read in the Dairy Chemistry book that alpha casein has the largest number of hydrophobic regions than the other beta and kappa caseins. It's really funny. Beta casein works, but not that consistently well. Kappa casein is basically useless and alpha casein works nicely. There is a relation here.
- Whole casein requires heat and constant stirring to get it into solution.
- Alpha casein takes any where from 60-80 minutes of constant stirring at room temperature to get it into solution.
- Beta casein goes into solution in about 30-40 minutes.
- Kappa casein goes into solution the easiest at 15-20 minutes.
- Whole casein supports the greatest number of microtubules for the gliding motility assay.
- Alpha casein does an equally good job of supporting motility.
- Beta casein isn't as good as alpha casein.
- Kappa casein is just too inconsistent to be useful.
Not to mention there are some rather odd caveats to using beta and kappa casein. I have not tried using the slide warmer with alpha casein but I have a suspicion that it will work. However, using it with beta casein causes the assay to fail.
This is exceedingly odd. It would seem that the caseins that work the best at passivating surfaces for gliding motility assays are the ones that are the most difficult to get into solution. Does that mean they are the ones with the most hydrophobic regions? If that the case, then there is something very interesting and physical going on here. Although, a big issue with that statement is that the prevailing theory about casein micelle structure is that the kappa casein is on the surface of the micelle. This allows the micelle to stay in solution because it has a large hydrophilic area due to the fact that it is a glycoprotein. But, it is super tough to get into solution. Wouldn't a structure with a hydrophilic surface want to go into solution easily?
Moving forward, alpha casein 2nd try
Keeping up the string of experiments, I'm going to do all caseins 3 times. Then I should have a story to tell.
Right of the bat, things are looking great with this assay.
- Movie 2
- There is a MT with a huge Taxol clump on its minus end.
Beta casein 2nd try
Again, I am having to be near the tape to get any sort of motility going.
Unfortunately, I think I'm going to have to stop this assay due to the fact that my MTs are fading. I need to make a new motility solution. And, there's always tomorrow...