User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/05/25/More on kinesin speed effects due to temperature
So, yesterday I looked at a temperature study on the speed of kinesin due to temperature affects. This is probably going to be my last investigation into this since it is clear that there is an issue when taking my measurements that needs to be fixed by temperature stabilization.
I suppose I should return to this at some point in time since it is very interesting and doing a quick literature search shows that there are some interesting ideas out there.
It was brought to my attention that the microscope slide I am currently using may not be allowing for the most optimal amount of heat transfer when illuminated. So, I am going to look at how things change using a 100µm thick coverslip as the support. I am going to do the same procedure for the flow cell that I always do.
I am going to prepare a slide as usual and start taking data immediately as always. Along side of this experiment, I am going to run another one. This experiment will use a normal flow cell and instead of taking data immediately, I am going to wait till the first experiment is done and then look at the second. This is to look at the initial low data point I always get when running experiments with alpha casein. If there is no low point, then the affect I am seeing is not due to temperature. What it is, still needs to be determined.
Well...I ended up doing a lot of assays.
Experiment 1: I used a cover slip in place of my slide to see if the glass that I make my flow cells with is too thick thus causing a thermal insulation when taking data.
Experiment 2: Using a regular flow cell I did experiment 1 and left experiment 2 on the bench. When I was done taking data for experiment 1, I started taking data for experiment 2. This experiment is to check and see if the initial data point for alpha casein passivation is due to temperature. If it's still there in this data, then it's something else.
Experiment 3: Koch suggested I heat the microscope slide and look at speeds. Presumably, the microtubules should speed up when I put a block of aluminum on the slide that was heated in a dry bath to 38˚C. They should then decrease in speed when I took the block off.
Experiment 4: I want to see if the reason why the speeds are so slow in the beginning of a data run is due to the kinesin. When kinesin walks "off" the microtubule in the assay, it doesn't know it is coming to the end of the MT. Thus, its motor domains are in a state of "ready for another MT" with one head having a different nucleotide state than the other one. As opposed to initially in the experiment when the kinesin has both its motor domains with ATP bound to them. I think that one reason why we see a speed up is because kinesin is in a non equal nucleotide state later on. I'm running a sample in a regular flow cell with alpha casein passivation for 1 hour total illumination time. That way, if we track a MT going over one region of kinesin, then there is a higher probability of finding another MT in that time frame that will go over a similar if not the same region. My hope is to see a speed up but I think temperature effects will probably dominate.
So I was able to run experiment 1. It would appear that the glass slide in fact does cause a good bit of thermal insulation from the microscope. This can be seen from the fact that the initial low data point that I typically see in an alpha casein passivation study isn't there.
This data is a bit odd. I wasn't able to measure the initial 5 data points because the software isn't allowing me to for some reason. However, there could be an increase in temperature or, it could just be an illusion. The line is the average value of the data points. I can't really say anything about temperature due to the fact that I'm missing the first 5 data points. The data is faster than the above data from experiment 1.
Yep. Heating up the sample with the aluminum block increased the speed of the MTs. The removal of the block then slowed them down as is seen in the following graph.
I can't analyze this data since it is one big file and not broken up into smaller ones. The movie is cool though.
After thinking about this some more, I'm not sure my setup as it is right now is sensitive enough to determine my initial reason for doing this. I think the best way to observe what I think is going on is to do single molecule, optical tweezer work.
There are a few very important things to discuss.
- The increase in speeds I was seeing over time is definitely due to a temperature affect.
- The casein studies I did previously may not be correct in that their speed differences may not be real due to the timing of the observations.
- The unusual dip in my D2O data may be due to a temperature affect as well since the last few data points were taken at a later date with the Hg lamp being off between experiments.
- I turned on the Hg lamp today and measured the temperature of the objective. In 4 hours time the temperature went from around 24˚C to 25.6˚C and it was still going up when I left. I've left it on over night and I'll check the temp again in the morning.
Erik is completely correct when he states that it takes a long time for things to stabilize with temperature. I am indebted to him for his intuitive help. And, for his kick ass temperature stabilization ideas.
Andy Maloney 11:52, 27 May 2010 (EDT): Update: I have left the Hg lamp on for about 24 hours. The temperature on the objective is finally holding steady at 25.3˚C. I would also like to point out that at 6% Hg illumination, the temperature at the center of the objective is around 27.3˚C which is significantly higher than room temperature. Looks like I can use the Hg lamp with temperature stabilization of the objective if I heat it above 27˚C.