User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/04/01/Casein assay 2
So at the beginning of last month I was able to complete my casein assay study. Here, I repeat the experiment to ensure reproducibility.
I was only capable of doing 3 of the caseins for the first day of data taking. These were done on Tuesday and I have the automation software cranking along right now to track the data.
So for each of the casein assays I did the following:
- Measured the time from sealing the slide to the first ROI where I start to take data. I should point out that after focusing the microscope, I positioned the sample so that the objective was in the middle of it. I then moved the slide such that the next FOV was approximately 2 FOVs over from the previous one. This way I stay relatively close to the center of the slide chamber for all my regions. I also periodically check to make sure I'm taking data no where close to the edges of the flow cell.
- I did take data beginning from the tape of each assay. This is to investigate how the edges of the flow cell may play a role in the speed at which microtubules glide.
- The microscope was setup such that the FOV in the camera is the only place illuminated in the sample. I also used a 6% Hg transmission filter effectively reducing the power of the Hg lamp by more than 94%, since I have a filter cube etc. The gain in the camera software was set to 150 and the exposure time is set to 100 ms.
- I took 15 ROI. Each ROI has 600 frames for a total illumination time of approximately 2 minutes.
- For the regions close to the tape, I positioned the objective such that a portion of the tape was visible in the FOV for the first measurement. I then moved the objective away from the tape for the next ROI. Each ROI was a full FOV away from the previous one. I took 5 total regions at the same frame rate as my regular regions.
First 3 caseins, whole, α, and β
So for this experiment I did the following:
- I polymerized microtubules.
- I got fresh aliquots of:
- I made fresh solutions and suspensions of:
- Make pages for the different casein solutions
- Again, I polymerized microtubules.
- I used the aliquots of PEM-A, PEM-Glu, and kinesin from Tuesday.
- I made a fresh batch of Antifade.
For this experiment I again did my usual 15 ROIs. I also looked at the tape affects. Unfortunately, there is just not a lot of microtubules that get on the kappa casein passivation. So I had to hunt all over the place for both the tape and regular ROIs. I did maintain a good deal of distance from the tape in my regular ROIs.
I should note that I didn't allocate my time properly so the kinesin in the flow cell incubated for 10 minutes instead of the usual 5. Update the Kinesin page. I didn't notice anything unusual in the sample so I believe that the extra time for incubation is not a problem. We will see though when automation is finished.