User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/08/07/Passivation study

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Introduction

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.

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