User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/08/06/Casein studies

Whole casein 1st try
So when I went to start taking data, the first 4 movies are useless due to the fact that most if not all the MTs in the FOV broke apart. I'm not sure what the cause of this is as I prepared a new stock solution of Taxol. It could be due to my Antifade not working. However, the MTs that are in the FOV do not dim, they just break apart.

After the first 4 movies, everything seems to be working normally.
 * Steve Koch 18:52, 6 August 2010 (EDT): This is a classic problem from when I was around these assays at Sandia. I don't know the cause.  Antifade is a suspect, even if you don't see photobleaching.  There is that paper Haiqing sent us that uses Ascorbic acid to prevent this.  Too bad it's happening...anything you're using a new batch or lot?
 * Andy Maloney 19:42, 6 August 2010 (EDT): Batch. Made when I remade everything for the first time. Beats me as to what is going on. I'm just going to remake it. I really hate this antifade stuff. Maybe I should switch to either the vitamin C or vitamin E alternatives. The big question would be; How much to use? Right now the antifade system constitutes a lot of osmotic pressure. What will happen if I end up using only 100um Trolox as my antifade system? Or, do I use at least 200 mM of it since that's the amount of glucose used in the antifade system.

Beta casein 1st try
So, as before, there is no motility. I'm using 1mg/mL beta casein in PEM to passivate the slide for 10 minutes. I then flow in 0.5mg/mL beta casein in PEM with 27ug/mL kinesin and 1mM ATP. No motility.

Now I'm questioning if the slide warmer is doing this. It's almost as if there is no kinesin on the slide at all.

Beta casein 2nd try

 * Again. 1mg/mL beta casein in PEM for 10 minutes.
 * 27ug/mL kinesin + 1mM ATP + 0.5mg/mL beta casein in PEM for 5 minutes. Added kinesin to solution 1 minute before add to slide.
 * Add motility solution.
 * No slide warmer.


 * First movie
 * Okay this is just weird. I see motility now. Although not as much as the whole casein study. And, they aren't really staying motile. Hmm.
 * I can't take any more as there is nothing moving around.

I'm flummoxed! I swear I got this to work before. Maybe a new batch of kinesin.

Beta casein third try

 * 1.0mg/mL beta casein in PEM for 10 minutes
 * 0.5mg/mL beta casein in PEM + 1mM ATP + 27ug/mL kinesin for 5 minutes.
 * Motility solution.
 * No slide warmer and new kinesin.

Okay, perhaps it was the kinesin. I have motility going on but, I'm still getting lots of opticution. Maybe it's my antifade. The first movie showed a lot of it. The second, not so much.

Ha! This was the third try...

I'm curious to see at what point in time the speed levels off. If it is within the first 5-7 data points, then using the slide warmer may not be beneficial at all.

Some good news about this assay is that it is behaving as the whole casein assay did with the opticution. The first 4 movies all end in opticution. The odd thing is that it is still going on in this assay. It didn't stop till the 9th movie but, it could have been the area I was in on the slide. Things seem to be going much more smoothly now.

Beta casein fourth try

 * 1mg/mL beta casein in PEM for 10 minutes
 * 0.5mg/mL beta casein in PEM + 1mM ATP + 27ug/mL kinesin for 5 minutes.
 * Motility solution.
 * New antifade.
 * Slide warmer.

If I don't get motility with the slide warmer, then I just can't use it for the casein constituents experiments.

I should note that this is a new motility solution with new antifade. I really hate the antifade system! I did see a paper that uses the oxygen scavenging stuff plus Trolox. Trolox is an antioxidant. BME is stinky. I think I'm going to try Trolox in place of the BME sometime in the future.

Ha! One can get motility using the slide warmer but, not as much as I've seen without it and it took 10 minutes for me to find mobile MTs. I'll have to look at the data with the beta casein passivation without using the slide warmer. If it turns out that I can get good data without it, it's getting '86-ed.

Also, there is no more catastrophes going on. So, it was the antifade. I did come across an article that says the BME in our antifade system is just used to stop Cy3 or Cy5 from blinking. To me, this means I don't need the crap.

Also, the MTs are gigantic which means that there is not enough kinesin on the surface to support small MT motility. Hmm. Does this mean that cooling the slide will result in more adhesion of the casein which will then have more kinesin on the surface? Sounds like trial 5...

Beta casein fifth try

 * 1mg/mL beta casein in PEM for 10 minutes.
 * 0.5mg/mL beta casein in PEM + 1mM ATP + 27ug/mL kinesin for 5 minutes.
 * Motility solution.
 * I found a piece of aluminum in the fridge so I'm passivating the slide on that. Just to see if temp is the issue with the passivation.

Problem solved!
Oh Yeah! That's the ticket!

So with out a doubt in my mind this is the issue going on. In the process of stabilizing the temperature for the objective, I made a slide warmer. The reason why we want a stable temperature for the objective is to be able to get steady speed data for the gliding motility assays. I thought that in order to get the best possible speed measurements, I needed to warm the slide while I passivated the surface and added kinesin. The reasoning behind this was to ensure that when I put the slide on the microscope, the temperature would 1, not decrease too much and thus cause temperature spikes from the controller, and 2, I reasoned that it would just be better all around for data taking. Since the slide would be close to the temperature of the objective, I wouldn't have to take non stable speed measurements. But, by warming the slide during incubation, I prevented the casein from adhering to the glass and thus not allowing the kinesin to adhere properly for the gliding motility assay to work.

This has been a rather long road to determine this. I first thought that my chemicals went bad when I tried an assay using the slide warmer and the temperature stabilized objective. So, I remade all my solutions. This probably was not necessary but I didn't know it at the time. I assumed that since nothing was working, my chemicals went bad because the assay was behaving as if that was the case.

I didn't write down my procedures when I initially made my solutions, so I was making them blind the second time around. I made them and filtered them through a 200nm filter. I tried my assays and guess what, they still didn't work. I then thought that the reason as to why things stopped working was because of my filtering. Since I didn't have notes saying that I did this step or not when I first made the solutions. On top of this, I was using the incorrect whole casein to make one solution. I tried 3 times to get the whole casein to go into solution to no avail. Then I realized that it was the wrong chemical. That wasted 3 days of my time. It's a good lesson though. I'll always write in my notebook everything I do.

So, after getting the whole casein to go into solution, I tried my assays again. Nothing worked so I remade everything again only this time I increased the concentration of the caseins in solution and I didn't filter them. I did this because I was able to determine that the reason nothing was working was because I didn't have enough casein on the glass surface. So, I reasoned that the filter was removing all my casein from solution. Not sure about this one but I thought it.

Now, after remaking everything for the third time, the final piece to my temperature stabilized objective arrived and I put it in place. It turns out that what I thought the temperature on the top of the objective was, was wrong. This was because of an improper measurement device. So, when I looked at the actual temperature, I noticed that I was taking data at 32C instead of what I though which was 30C. So, I retried my experiment with some whole casein passivation, which by the way is not sensitive to being warmed up before going on the objective. It turns out that 30C is too low of a temperature with my current setup and recent data showed that the speed measurements increased over time. This lead me to today.

After figuring out that I needed to be at or above 32C to take data, I tried my assays again, at 33C. Again, nothing worked except the whole casein assay above. Through a process of elimination, I was able to determine that the reason why nothing worked was because of the warming of my slides during incubation. I realized this after I took a hunk of aluminum that was randomly in the 4C fridge and passivated the surface of a slide while it was on this cold block. I am right now taking data that looks spectacular since I was able to get proper adhesion of the beta casein to the slide which in turn allowed for proper kinesin adhesion.

My plan is to take the data out to at least 25, 2 minute ROIs. This way I can see where the gliding speeds level off and to see if I can get a good measurement from the data. If it turns out I don't need that many data points, then great. If I need more, then I'll do more. The important thing however is that I was able to solve my problem!

Another good thing that I learned is that there is a possibility that I don't need to use BME for my antifade any more. That would be spectacular and is something that I will look into.