User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/08/11/More caseins
So, to ensure that all my chemicals and assay are working properly, I am doing an assay using the alpha casein passivator. So far, everything looks great and I will be able to move on to the kappa and beta passivation.
But, since I'm here and I have the assay running, I'll go ahead and take the data for a normal run.
- Movie 4
- There is a big chunk of gunk stuck on a microtubule. Ha! I like the alliteration.
Kappa casein 2nd try
And, no change from the other day. Very little motility and lots of stuck guys.
For fun...M casein
I decided to mix up "whole" casein by mixing alpha, beta, and kappa in the following proportions:
- 49% alpha
- 37% beta
- 14% kappa
These are approximately the same values as is quoted in Dairy chemistry and biochemistry which has them at:
- 47% alpha
- 35% beta
- 12% kappa
with I'm guessing 6% extra stuff. I'm going to call my solution M-PEM since it's a mix.
So the as before, just with this solution.
- 1mg/mL mixed whole casein in PEM for 10 minutes.
- 0.5mg/mL mixed whole casein in PEM + 1mM ATP + 27ug/mL kinesin for 5 minutes.
- Incubation done at room temperature.
Andy Maloney 14:11, 11 August 2010 (EDT): I'm going to give this a 90% chance of supporting motility. I'll see if I'm right.
Andy Maloney 14:22, 11 August 2010 (EDT): Ha! It's working splendidly.
Although, I'm rather disappointed with the number of Taxol crystals I have in solution. I'm guessing I had a lot of free tubulin after polymerizing the microtubules and this is causing them to aggregate. I'll make another batch of microtubules after this run.
Beta casein final assay
So this is the final assay that I'm going to do with beta casein. It's not that reliable but I am getting some motility.
I should also note that I didn't polymerize more microtubules, I just made a new motility solution and yes, there are still Taxol crystals in it.
Great. Now everything is disintegrating. No...that was too close to the tape.
Also, I am unable to get any data from this. There is just not enough MTs on the slide for it to be worth anything.
Well, the beta casein and kappa casein just don't work. Plus, I was getting way too many Taxol crystals in my motility solution and I think it is due to an uncalibrated pipettor. (Started using a different one very recently for the Taxol.)
I have to make flow cells, more antifade and autoclave stuff so I have to stop experiments today.
I hate BME
I mean I really hate it. So, I decided to try a motility assay with alpha casein passivation and no BME in my antifade. Well, I'm not getting any motility and since the MTs are stuck in one place, they are breaking up. Looks like I need the crap for this experiment to work properly.
Maybe the MTs break up because of reactive oxygen. I can try adding Trolox to the antifade mix to see if that help because right now, it won't work. This will have to be a side project because I really don't have the time to work on it.