User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/29/Beta casein test
Yesterday, I found out that none of my casein solutions were supporting motility except for my whole casein. I have narrowed the reasons for this down to the possibility that it was storing them in the -80C freezer. Today, I prepared more beta-PEM and I'm going to try an assay to see if it works. I did send the solution through a 0.2um filter.
Nope...No motility on the slip. I did see a small amount on the slide but I can't use that. I'll wait a few more minutes to see if anything is working.
15 minutes in and still nothing.
I don't get it. It just isn't working. And of course I didn't save some beta-PEM to not filter it so I could see if that was the issue.
- The Taxol is working just fine because the MTs are stable.
- The Antifade is working because after blasting them with 100% illumination, they are still bright.
- The kinesin is good as has been shown with the whole casein assay. As well as the PEM-Glu and PEM-A are good because whole casein works.
- The freezer isn't the problem as was shown right now.
- The filtering can't be the problem because the whole casein works. Unless the filter is gobbling up all the casein. But then I wouldn't see bubbles forming in my casein solutions, which I do so there is protein in them.
This truly has become a mystery now. Maybe if I get a new batch of kinesin I'll try this again. But, I'm out of ideas and there is no point trying to brute force anything now.
After 1.5 hours, nothing.
I know I said I would stop but I did it again anyway. This time without filtering the beta casein solution. The assay works.
Again, I find that I am overwhelmingly infuriated at myself for not writing this down earlier. All this trouble is because of my lack of notebook entries.
2 weeks waisted! I will say however that the assay seems to be a little scarce on MTs gliding. It may be because I'm using older MTs but I don't think so. I am having to hunt for MTs that are capable of being tracked and that are not near the edges of my flow cell. So, there is still something wrong with the assay but at least I have it working again.
There does seem to be a higher density of MTs gliding on the slide than they are on the slip. I know I had said that it couldn't be the slide warmer because my whole casein works fine but now I have to question if it is now that I know filtering casein constituents is not a good idea.
Okay. Here's the setup.
- beta-PEM, unfiltered and unfrozen.
- No slide warmer.
- Same prep as usual.
There are still a lot fewer MTs gliding than I had in previous experiments but, this is the best so far. I have MTs gliding on the slip now in the very least. And, they are remarkably long. I'm talking 50um long.
I'm not sure what is going on here but I am getting a lot more motile MTs. I'm curious if I was inadvertently using a higher concentration of beta casein in my assays before hand.
I'm going to use unfiltered beta-PEM and no heating pad on the slide. I'm also going to passivate the surface with 0.5mg/mL beta-PEM but also use 0.5mg/mL beta-PEM when I introduce the kinesin to the slide.
Well, it turns out that this works. Lots of MTs on the slip and small ones are exhibiting motility. This assay is working well enough where I believe I will be able to get enough data for an entire run. Great!
Now, I'm super curious to see if the filtered stuff is okay and that all I need to do to use it is have the higher concentration on my slides.
There is one good thing that is coming out of this and that's the fact that I now know the characteristics of an assay that does not have enough surface passivation on it.
Okay. So here goes again. This time I will be using:
- The frozen and filtered beta-PEM that has 0.5mg/mL beta casein in it.
- I will passivate my surface with beta-PEM, which has 0.5mg/mL beta casein in it, for 10 minutes.
- I will then add kinesin in beta-PEM and 1mM ATP and let this incubate for 5 minutes.
- I will then flow in MTs.
- All of this will be done on the hot plate.
So no dice with this one. Although, it does have the characteristics of there not being enough casein in solution to support motility.
I'm going to freeze some of my non filtered beta casein and see if it's the freezing that is causing issues with the casein.
And the answer to that is no. It's the filtering. I've motility working just fine.