User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/28/Caseins

Beta casein

First attempt

So I prepared a slide as usual with beta casein passivation. However, it has been 13 minutes since I removed the slide from the hot plate to the microscope and I still am not able to see motility. This could mean that my kinesin is bad but, I used this same kinesin yesterday to look at whole casein passivation. I'm not sure what is going on. I'll keep it on the microscope for a little longer to see if motility occurs. If it doesn't, then I'll have to try a different vial of kinesin.

After 30 minutes, still nothing. Now, I cannot remember when I first did the beta casein solution, if I filtered it through a 0.2um filter or not. And of course, I didn't write the damn thing down in my notebook. I have no clue as to if this is the reason why my assay is not working or not but it may be. I'm going to try a different vial of kinesin first as this is the easiest thing to do. If is still doesn't work, then I'll try making another solution of 0.5mg/mL beta casein in PEM but I won't filter it.

Second attempt

The second attempt I tried a new aliquot of kinesin. This did seem to work however, with a caveat. I'm only seeing motility on the slide and not on the slip. Trying to track microtubules on the slide is going to be impossible due to the massive amount of noise caused by the other microtubules in solution.

I did take a look at my first attempt again to see if there was any motility going on on the slide. There was none which makes me believe that the kinesin I have just doesn't want to stick to beta casein. This is a new phenomenon.

Returning to slide #2 shows that there is still a small amount of motility going on on the slide.

Now I don't know if the reason this is occurring is due to something I did differently when preparing the flow cell of not. I'm going to make a new flow cell not from my stock to see if this is the case.

Third attempt

So I made a fresh flow cell and still I couldn't get it to work. The reason why I made a new one is because when I made the stock batch that I have now, I cleaned the slides when previously, I didn't. I used the new aliquot of kinesin and still used the hot plate at 30C. No motility.

This is exceptionally odd since I know I was able to get this to work 6 months ago and I know I filtered the beta casein PEM solution just like I did this time around. I just don't get it. The whole casein passivation works.

Alpha casein

I'm not sure what is going on with beta casein surface passivation. It just seems to have stopped working for me and I have no clue as to why. It may be because of the kinesin but I'm not sure. At any rate, I'm going to try the alpha casein passivation now.

I don't know if this is an issue but I did freeze all my casein solutions in the -80C freezer. The beta casein didn't work and now I'm going to try the alpha casein. If it doesn't work, then I'm going to try the whole casein again from the freezer. If that doesn't work then I know it's the freezer's fault. However, if the alpha casein works then I think there is something wrong with my kinesin.

First attempt

I see no motility using an alpha casein passivation. Again, I'm going to wait a bit to see if anything happens but it doesn't look good.

Here's what I know so far:

• Cleaned slides are not the issue.
• It's not just the beta casein.

The following could be issues.

• The slide warmer.
• The freezer.
• Filtering.
• It's the kinesin.

If the freezer is the cause, then the whole casein should not work. Although there could be big differences between the constituents of casein and whole casein and how they behave under extreme temperatures.

I know it isn't the slide warmer since I've tried that already and still I saw no motility. I guess I'll try the whole casein from the freezer now.