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User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/30

Andy Maloney — Fri, 30 Jul 2010 14:31:43 GMT

Entry title:

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|style="background-color: #EEE"|[[Image:Chef2.png|128px]]<span style="font-size:22px;"> Lab notebook of Andy Maloney
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|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>      </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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|colspan="2"|<html><input type="radio"><a href="http://www.openwetware.org/index.php?title=User:Andy_Maloney/Kinesin_%26_Microtubule_Page/PEM_buffer&oldid=369093">PEM</a></input></html>
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==New (to me) technology==
So Google has done a pretty spectacular thing. It allows me to upload the movies I generate from my experiments to Google Docs. It then allows me to have the entire contents of an experiment (in avi form) available to everyone. For instance, here is a folder containing an assay,

https://docs.google.com/leaf?id=0B4rJrKuZEFPWYThlYzYyOGItODBmOS00Y2RlLWE4ZTMtZmIzYjIwZWY1ODc0&hl=en

It works quite well. The only issue I have with it is that you are not able to preview the movie file in Google Docs. I'm sure this will change at some point in time but for now it works great.

On top of the spectacular ability to upload my files, I can tag them using the xmp protocols from Adobe. And guess what, we have the Creative Suite to tag my files! This means I can have metadata attached to my videos! I can then search my videos via Google and have the capability to download them any where. This is awesome. Of course, it is going to be a massive amount of work considering I have nearly 0.5TB worth of data to tag. But, I think it will be helpful for myself and anyone looking for my data in the long run.

Man, open science and technology kicks ass!


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User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/29/Beta casein test

Andy Maloney — Thu, 29 Jul 2010 23:50:23 GMT

Fourth try:

New page

{{AndyMaloneyNotebook
|Description=Trying the beta casein assay without putting it in the freezer and without filtering it and not putting it in the freezer. Guess what, it was the filtering.
}}
[[Category:Kinesin and microtubules]]
[[Category:Surface passivation]]
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.
==First try==
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.
==Second try==
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.
==Third try==
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.
==Fourth try==
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.
==Fifth try==
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.
==Sixth try==
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.

User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/29

Andy Maloney — Thu, 29 Jul 2010 16:34:02 GMT

Entry title:

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User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/26/Microscope repair

Andy Maloney — Thu, 29 Jul 2010 04:40:49 GMT

The thing that broke:

←Older revision		Revision as of 04:40, 29 July 2010
Line 15:		Line 15:
	In fact, you shouldn't read any further and you should just call a technician to come and fix/clean your microscope. These are my notes and you shouldn't use them.		In fact, you shouldn't read any further and you should just call a technician to come and fix/clean your microscope. These are my notes and you shouldn't use them.
	==The problem==		==The problem==
-	When diverting the light path from the eyepiece to the camera, you use a knob on the microscope ~~to move some~~ optics ~~that diverts the light path~~. This knob broke 3 times on my microscope in 3 different places. Each time it broke, it cemented my belief that you should build your own microscope and just use the killer objectives from the microscope companies.	+	When diverting the light path from the eyepiece to the camera, you use a knob on the microscope that moves optics. This knob broke 3 times on my microscope in 3 different places. Each time it broke, it cemented my belief that you should build your own microscope and just use the killer objectives from the microscope companies. That way when something breaks, You know how to fix it easily.
-	~~==Tear down==~~	+
-	~~The first thing I did is look at the nice pretty microscope we just bought with the awesome funding we received. Then~~, ~~I realized that I had~~ to ~~take~~ it ~~apart and fix poor quality control~~.	+

		+	==Tear down==
	<center>		<center>
	[[Image:AM_IX71Built.JPG\|400px]]		[[Image:AM_IX71Built.JPG\|400px]]
	</center>		</center>

-	We do not have a camera attached to the microscope. We placed the camera on a 3 axis stage mounted on the table in order to reduce vibrations on the microscope. It's not necessary but it sure does make life a heck of a lot easier to position the camera in the center of the field apertures. I'd recommend it to everyone as it reduces the area you have to illuminate and thus reduces photobleaching of the sample. So why is it then that the field aperture isn't centered on the camera already? Well, there is not enough degrees of freedom to center the Hg lamp aperture on the camera due to how the lamp mounts on the microscope. So even if an engineer was able to center it in the factory, you're going to have to replace that Hg bulb at some point in time and you'll never get it in the same place again.	+	We do not have a camera attached to the microscope. We placed the camera on a 3 axis stage mounted on the table in order to reduce vibrations on the microscope. It's not necessary but it sure does make life a heck of a lot easier to position the camera in the center of the field apertures. I'd recommend it to everyone as it reduces the area you have to illuminate and thus reduces photobleaching of the sample. So why is it then that the field aperture isn't centered on the camera already? Well, there is not enough degrees of freedom to center the Hg lamp aperture on the camera due to how the lamp mounts on the microscope. So even if an engineer was able to center it in the factory, you're going to have to replace that Hg bulb at some point in time and you'll never get it in the same place again. Plus, it is super easy to place the camera focal plane at the eyepiece focal plane with a stage.
	===Hg Lamp Removal===		===Hg Lamp Removal===
	The first thing I did is take out the aperture lever. This just screws out of it's housing. I made sure I unplugged everything as well.		The first thing I did is take out the aperture lever. This just screws out of it's housing. I made sure I unplugged everything as well.
Line 31:		Line 30:
	</center>		</center>

-	Using the supplied hex wrench, I loosened the 2 set screws on the left and right of the microscope. I don't know what the set screw sizes are but I do know that the hex wrench is ~~an M4~~.	+	Using the supplied hex wrench, I loosened the 2 set screws on the left and right of the microscope. I don't know what the set screw sizes are but I do know that the hex wrench is 4mm.

	<center>		<center>
Line 70:		Line 69:

	<center>		<center>
-	~~Picture~~	+	[[Image:AM_IX71FilterTurretRemoval.jpg\|400px]]
	</center>		</center>
		+
	===Objective Turret Removal===		===Objective Turret Removal===
	I then removed the objective turret. There are 3 M5 screws that I loosened and made sure to keep them in the turret so as to not loose them.		I then removed the objective turret. There are 3 M5 screws that I loosened and made sure to keep them in the turret so as to not loose them.
Line 89:		Line 89:
	'''Front Plate Removal'''		'''Front Plate Removal'''

-	The first step is to remove the actual knob from the microscope. ~~There are 2 set screws holding it on a steel rod. Now I said that it was~~ this knob ~~that broke but in fact it~~ is ~~an internal piece~~ attached to ~~this~~ knob ~~that kept breaking on me~~.	+	The first step is to remove the actual knob from the microscope. In this picture, the knob is still attached to the microscope. When it broke, the knob plus a long metal post came out of the microscope.

	<center>		<center>
Line 113:		Line 113:
	</center>		</center>

-	With the front plate removed, I could see now where the screws were that kept the rest of the eyepiece attached to the microscope.	+	With the front plate removed, I could see now where the screws were that kept the rest of the eyepiece attached to the microscope. You can also see clearly in this picture the metal post that attaches to the knob I took off earlier.

	<center>		<center>
Line 132:		Line 132:
	[[Image:AM_IX71EyePieceLightCollector02.JPG\|400px]]		[[Image:AM_IX71EyePieceLightCollector02.JPG\|400px]]
	</center>		</center>
		+
	===Magnifier Removal===		===Magnifier Removal===
	The next part I removed, although in hindsight was unnecessary to get at the broken part, is what I'm calling the magnifier. I call it this since I can slide out a lever that moves an optic in and out of the light path that compound magnifies my image by 1.6X. At any rate, I took out 6 screws. 4 require a PH000 screwdriver and the other 2 require (I believe) a 2mm Hex.		The next part I removed, although in hindsight was unnecessary to get at the broken part, is what I'm calling the magnifier. I call it this since I can slide out a lever that moves an optic in and out of the light path that compound magnifies my image by 1.6X. At any rate, I took out 6 screws. 4 require a PH000 screwdriver and the other 2 require (I believe) a 2mm Hex.
Line 162:		Line 163:
	[[Image:AM_IX71MagnifierRemoval04.JPG\|400px]]		[[Image:AM_IX71MagnifierRemoval04.JPG\|400px]]
	</center>		</center>
		+
	==The thing that broke==		==The thing that broke==
-	So what kept breaking on me was an adapter piece. This adapter mated the ~~knob~~ that ~~moved~~ the ~~optic that diverted~~ the ~~light path from the eyepiece to the camera,~~ to a gear. To reach the piece, I had to remove the gearbox. The first time I took apart the microscope, I didn't take the gearbox out because I was afraid I was going to break it. This caused a tremendous amount of pain for myself since I was not able to reach the adapter piece that broke. At least not very easily. I spent a week working around the gearbox trying to reach the set screws on the adapter piece which eventually lead to the piece breaking in half. This is a lesson I've learned multiple times. If you see that it's screwed in, it's more than likely supposed to come out without much pain. As opposed to when something is glued in place.	+	So what kept breaking on me was an adapter piece. This adapter mated the metal post that was connected to the knob on the front to a gear. To reach the piece, I had to remove the gearbox. The first time I took apart the microscope, I didn't take the gearbox out because I was afraid I was going to break it. This caused a tremendous amount of pain for myself since I was not able to reach the adapter piece that broke. At least not very easily. I spent a week working around the gearbox trying to reach the set screws on the adapter piece which eventually lead to the piece breaking in half due to excessive movement on my part. This is a lesson I've learned multiple times. If you see that it's screwed in, it's more than likely supposed to come out without much pain. As opposed to when something is glued in place.

-	To take the gearbox out, I just loosened the 4 screws holding it in place~~. I place the microscope on it's back to get at it's bottom~~.	+	To take the gearbox out, I just loosened the 4 screws holding it in place.

	<center>		<center>
Line 171:		Line 173:
	</center>		</center>

-	I will have to say that when I went to put the gearbox back in the rebuild, I had to hold down the focusing platform while I screwed in the gearbox. ~~This way~~ the focusing ~~platform had its complete~~ range of motion.	+	I will have to say that when I went to put the gearbox back in the rebuild, I had to hold down the focusing platform while I screwed in the gearbox. When I didn't do this, I noticed that the focusing knobs did not have their full range of motion.

	<center>		<center>
Line 183:		Line 185:
	</center>		</center>

-	There are 4 set screws on this adapter, it's the black thing deep in the microscope. The top 2 hold the post that is connected to the light path diverter knob on the outside of the microscope. The bottom 2 hold the adapter and post to a gear that moves the optics. The first time this broke, the top 2 set screws came loose. The second time, the bottom 2 came loose. The third time, the adapter broke in half and I had to glue it together. To get to the set screws, I just approached them from the bottom of the microscope. I then moved the gears so that I could reach the other set screws.	+	There are 4 set screws on this adapter, it's the black thing deep in the microscope. The top 2 hold the post that is connected to the light path diverter knob on the outside of the microscope. The bottom 2 hold the adapter and post to a gear that moves the optics. The first time this broke, the top 2 set screws came loose. The second time, the bottom 2 came loose. The third time, the adapter broke in half and I had to glue it together. To get to the set screws, I just approached them from the bottom of the microscope. I then moved the gears so that I could reach the other set screws. I believe I used a 1.5mm hex screwdriver for the set screws.

	I can imagine that this piece will break for a great deal many people. If it does, call the technician to come fix it!		I can imagine that this piece will break for a great deal many people. If it does, call the technician to come fix it!
		+
	==Complete tear down==		==Complete tear down==
	After having to glue the adapter piece and having to tear down this thing a third time, I decided to take it down almost all the way. So I kept taking pieces off until the glue set. Here's what it looked like.		After having to glue the adapter piece and having to tear down this thing a third time, I decided to take it down almost all the way. So I kept taking pieces off until the glue set. Here's what it looked like.

User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/27/Reboot

Andy Maloney — Thu, 29 Jul 2010 03:38:52 GMT

←Older revision		Revision as of 03:38, 29 July 2010
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	==New solutions==		==New solutions==
	After remaking all of my expired solutions, I am running an experiment now. I'm using whole casein as the surface passivator since it is my baseline for data. Everything seems to be working fine and I'll just have to wait till the computer crunches the numbers before I can say if everything is working as I think it should.		After remaking all of my expired solutions, I am running an experiment now. I'm using whole casein as the surface passivator since it is my baseline for data. Everything seems to be working fine and I'll just have to wait till the computer crunches the numbers before I can say if everything is working as I think it should.
		+
		+
		+	[http://www.openwetware.org/wiki/User:Andy_Maloney/Notebook/Lab_Notebook_of_Andy_Maloney/2010/07/28/Updated_reboot Updated reboot]

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

Andy Maloney — Thu, 29 Jul 2010 03:32:26 GMT

First attempt:

←Older revision		Revision as of 03:32, 29 July 2010
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	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.		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.
	==Whole casein==		==Whole casein==
		+	So if this works, then I still won't know if it's the freezer or if it's the filtering. This will tell me if my kinesin is good or not and I suspect that it is since I was able to get good data yesterday. But, if it doesn't, then it's the freezing or filtering.
		+
		+	I also noticed that there is a lot of moisture in my bench top cooler. This may be causing contamination and I think the moisture came from the power outage recently. Not sure if this is an issue but it's probably a good idea for me to dry the beads out at any rate.
		+	===First attempt===
		+	It's working. I'm going to take a run of data and add it to yesterday's. So my kinesin is fine.
		+
		+	It would appear that freezing whole casein at -80C is okay as well as filtering it though a 0.2um syringe filter. I am 90% sure that in my earlier casein mixtures I passed them through a 0.2um syringe filter as well so I think it is the freezing that is causing the issue.
		+
		+	I wanted to freeze my casein solutions because I thought it would prolong their shelf life. There is a good thing out of all of this and that is that the assay I ran yesterday used whole casein as the passivation but, since my whole casein was prepared that day, I didn't put the aliquot I used in the freezer. If there is any differences in the speed at which microtubules glide at in this assay from yesterday, I know with better certainty that it's the freezing causing my issues.
		+
		+	I'd also like to point out that I'm seeing lots of very tiny microtubules gliding. This is an excellent indication that my kinesin is good.
		+
		+	[[User:Andy Maloney\|Andy Maloney]] 23:32, 28 July 2010 (EDT): The results are below.
		+
		+	<center>
		+	[[Image:AMWC2010_07_28.png\|400px]]
		+	</center>
		+
		+	Nothing odd going on here with the whole casein that was frozen in the -80C freezer. Yesterday's average was 925nm/s and today's average is 937nm/s. I'm assuming that the freezer must be causing the issues with the casein constituents not working as a passivator. I'm going to try tomorrow to make some beta casein without putting it in the freezer and see if I get motility. If I don't, then I'm abandoning this totally and I'm going to exclusively use the whole casein for all of my experiments with heavy waters.
		+
		+	==Bench top cooler==
		+	I have an unholy fear of contamination now thanks to a certain someone. This is why I evaporated all the moisture from my cooler. It has to cool down now and allows me time to analyze the data I just took to see if there is a speed difference between frozen and unfrozen W-PEM. It also gives me some time to figure out a way to stop moisture from collecting in my cooler.

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

Andy Maloney — Wed, 28 Jul 2010 18:06:31 GMT

←Older revision		Revision as of 18:06, 28 July 2010
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	==[[/Updated reboot\|Updated reboot]]==		==[[/Updated reboot\|Updated reboot]]==
	* Update from yesterday's reboot.		* Update from yesterday's reboot.
-	~~==[[/Beta casein attempt\|Beta casein attempt]]==~~
-	* With new solutions, I'm trying the beta casein passivation again.
-	~~==[[/Alpha casein attempt\|Alpha casein attempt]]==~~
-	* Trying the alpha casein passivation.
	==[[/Caseins\|Caseins]]==		==[[/Caseins\|Caseins]]==
	* Things aren't going very well.		* Things aren't going very well.

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

Andy Maloney — Wed, 28 Jul 2010 18:06:10 GMT

New page: {{AndyMaloneyNotebook |Description=Looking at casein passivation with all the new solutions made. Beta, alpha, whole so far. }} Category:Kinesin and microtubules [[Category:Surface pa...

New page

{{AndyMaloneyNotebook
|Description=Looking at casein passivation with all the new solutions made. Beta, alpha, whole so far.
}}
[[Category:Kinesin and microtubules]]
[[Category:Surface passivation]]
==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.
==Whole casein==

User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/28/Alpha casein attempt

Andy Maloney — Wed, 28 Jul 2010 18:06:07 GMT

Removing all content from page

←Older revision		Revision as of 18:06, 28 July 2010
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-	~~{{AndyMaloneyNotebook~~
-	~~\|Description=Trying out the alpha casein passivation with the new solutions. Since the beta casein isn't working for some reason, I'm moving on the the alpha casein.~~
-	}}
-	~~[[Category:Kinesin and microtubules]]~~
-	~~[[Category:Surface passivation]]~~
-	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.~~

User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/28/Beta casein attempt

Andy Maloney — Wed, 28 Jul 2010 18:04:52 GMT

Removing all content from page

←Older revision		Revision as of 18:04, 28 July 2010
Line 1:		Line 1:
-	~~{{AndyMaloneyNotebook~~
-	~~\|Description=Looking at the beta casein passivation with all the new solutions made.~~
-	}}
-	~~[[Category:Kinesin and microtubules]]~~
-	~~[[Category:Surface passivation]]~~
-	~~==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.

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

Andy Maloney — Wed, 28 Jul 2010 18:04:09 GMT

Alpha casein attempt:

←Older revision		Revision as of 18:04, 28 July 2010
Line 15:		Line 15:
	==[[/Alpha casein attempt\|Alpha casein attempt]]==		==[[/Alpha casein attempt\|Alpha casein attempt]]==
	* Trying the alpha casein passivation.		* Trying the alpha casein passivation.
		+	==[[/Caseins\|Caseins]]==
		+	* Things aren't going very well.

User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/28/Alpha casein attempt

Andy Maloney — Wed, 28 Jul 2010 18:03:29 GMT

First attempt:

New page

{{AndyMaloneyNotebook
|Description=Trying out the alpha casein passivation with the new solutions. Since the beta casein isn't working for some reason, I'm moving on the the alpha casein.
}}
[[Category:Kinesin and microtubules]]
[[Category:Surface passivation]]
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.

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

Andy Maloney — Wed, 28 Jul 2010 17:08:56 GMT

[/Alpha casein attempt|Alpha casein attempt]]:

←Older revision		Revision as of 17:08, 28 July 2010
Line 13:		Line 13:
	==[[/Beta casein attempt\|Beta casein attempt]]==		==[[/Beta casein attempt\|Beta casein attempt]]==
	* With new solutions, I'm trying the beta casein passivation again.		* With new solutions, I'm trying the beta casein passivation again.
		+	==[[/Alpha casein attempt\|Alpha casein attempt]]==
		+	* Trying the alpha casein passivation.

User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/28/Beta casein attempt

Andy Maloney — Wed, 28 Jul 2010 17:06:19 GMT

Second attempt:

←Older revision		Revision as of 17:06, 28 July 2010
Line 6:		Line 6:
	==First attempt==		==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.		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.

User:Andy Maloney/Kinesin & Microtubule Page

Andy Maloney — Wed, 28 Jul 2010 15:48:01 GMT

Buffers & solutions:

←Older revision		Revision as of 15:48, 28 July 2010
Line 22:		Line 22:
	* [[/PEM-T\|PEM-T]]		* [[/PEM-T\|PEM-T]]
	** This buffer is used to stabilize microtubules after polymerization.		** This buffer is used to stabilize microtubules after polymerization.
-	* [[/PEM-kC\|PEM-<math>\kappa</math>C]]
-	** Used to passivate the glass slides for motility assays. I like using [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=C0406\|SIGMA&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC <math>\kappa</math>-casein] because it goes into solution really easily.
	* [[/PEM-Glu\|PEM-Glu]]		* [[/PEM-Glu\|PEM-Glu]]
	** Used for the motility assay.		** Used for the motility assay.
Line 32:		Line 30:
	* [[/Antifade\|Antifade]]		* [[/Antifade\|Antifade]]
	** This is used as an oxygen scavenging system that aids in preventing photobleaching.		** This is used as an oxygen scavenging system that aids in preventing photobleaching.
		+	* [[/PEM-kC\|PEM-<math>\kappa</math>C]]
		+	** Used to passivate the glass slides for motility assays. I like using [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=C0406\|SIGMA&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC <math>\kappa</math>-casein] because it goes into solution really easily.
		+	* [[/W-PEM\|W-PEM]]
		+	** PEM with 0.5mg/mL whole casein in it.
		+	* [[/B-PEM\|<math>\beta</math>-PEM]]
		+	** PEM with 0.5mg/mL beta casein in it
		+	* [[/A-PEM\|<math>\alpha</math>-PEM]]
		+	** PEM with 0.5mg/mL alpha casein in it.
		+	* [[/K-PEM\|<math>\kappa</math>-PEM]]
		+	* PEM with 0.5mg/mL kappa casein in it.
		+
	=====Suspensions=====		=====Suspensions=====
	* [[/1 N NaOH\|1 N NaOH]]		* [[/1 N NaOH\|1 N NaOH]]

User:Andy Maloney/Kinesin & Microtubule Page/K-PEM

Andy Maloney — Wed, 28 Jul 2010 15:47:55 GMT

New page: ==Chemicals== ====[http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=C0406|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Kappa casein]==== ====[[User:Andy_Maloney/Kinesin_%26_...

New page

==Chemicals==
====[http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=C0406|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Kappa casein]====

====[[User:Andy_Maloney/Kinesin_%26_Microtubule_Page/PEM_buffer|PEM]]====
See the page for more information.
==Recipe==
* [[User:Andy_Maloney/Kinesin_%26_Microtubule_Page/PEM_buffer|PEM]]
* 0.5mg/mL kappa casein
==Notes==

User:Andy Maloney/Kinesin & Microtubule Page/A-PEM

Andy Maloney — Wed, 28 Jul 2010 15:47:18 GMT

New page: ==Chemicals== ====[http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=C6780|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPECi Alpha casein]==== ====[[User:Andy_Maloney/Kinesin_%26...

New page

==Chemicals==
====[http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=C6780|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPECi Alpha casein]====

====[[User:Andy_Maloney/Kinesin_%26_Microtubule_Page/PEM_buffer|PEM]]====
See the page for more information.
==Recipe==
* [[User:Andy_Maloney/Kinesin_%26_Microtubule_Page/PEM_buffer|PEM]]
* 0.5mg/mL alpha casein
==Notes==

User:Andy Maloney/Kinesin & Microtubule Page/B-PEM

Andy Maloney — Wed, 28 Jul 2010 15:46:18 GMT

New page: ==Chemicals== ====[http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=C6905|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Beta casein]==== ====[[User:Andy_Maloney/Kinesin_%26_M...

New page

==Chemicals==
====[http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=C6905|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Beta casein]====

====[[User:Andy_Maloney/Kinesin_%26_Microtubule_Page/PEM_buffer|PEM]]====
See the page for more information.
==Recipe==
* [[User:Andy_Maloney/Kinesin_%26_Microtubule_Page/PEM_buffer|PEM]]
* 0.5mg/mL beta casein
==Notes==

User:Andy Maloney/Kinesin & Microtubule Page/W-PEM

Andy Maloney — Wed, 28 Jul 2010 15:44:09 GMT

New page: ==Chemicals== ====[http://www.sigmaaldrich.com/catalog/Lookup.do?N5=All&N3=mode+matchpartialmax&N4=c7078&D7=0&D10=c7078&N1=S_ID&ST=RS&N25=0&F=PR Whole casein]==== ====[[User:Andy_Maloney/...

New page

==Chemicals==
====[http://www.sigmaaldrich.com/catalog/Lookup.do?N5=All&N3=mode+matchpartialmax&N4=c7078&D7=0&D10=c7078&N1=S_ID&ST=RS&N25=0&F=PR Whole casein]====

====[[User:Andy_Maloney/Kinesin_%26_Microtubule_Page/PEM_buffer|PEM]]====
See the page for more information.
==Recipe==
* [[User:Andy_Maloney/Kinesin_%26_Microtubule_Page/PEM_buffer|PEM]]
* 0.5mg/mL whole casein
==Notes==

User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/28/Beta casein attempt

Andy Maloney — Wed, 28 Jul 2010 15:34:18 GMT

New page: {{AndyMaloneyNotebook |Description=Looking at the beta casein passivation with all the new solutions made. }} Category:Kinesin and microtubules Category:Surface passivation ==Firs...

New page

{{AndyMaloneyNotebook
|Description=Looking at the beta casein passivation with all the new solutions made.
}}
[[Category:Kinesin and microtubules]]
[[Category:Surface passivation]]
==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.

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

Andy Maloney — Wed, 28 Jul 2010 15:31:02 GMT

Updated reboot:

←Older revision		Revision as of 15:31, 28 July 2010
Line 11:		Line 11:
	==[[/Updated reboot\|Updated reboot]]==		==[[/Updated reboot\|Updated reboot]]==
	* Update from yesterday's reboot.		* Update from yesterday's reboot.
		+	==[[/Beta casein attempt\|Beta casein attempt]]==
		+	* With new solutions, I'm trying the beta casein passivation again.

User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/28/Updated reboot

Andy Maloney — Wed, 28 Jul 2010 14:17:27 GMT

Data:

New page

{{AndyMaloneyNotebook
|Description=Data from yesterday's reboot.
}}
[[Category:Kinesin and microtubules]]
[[Category:Surface passivation]]
==Data==
Below is a graph of the data I took yesterday. It is with whole casein surface passivation. Everything seems to be working just fine and the speed at which I see motility is steady through the last 20 minutes of my data taking.

<center>
[[Image:AM_WCNewSolutions.png|400px]]
</center>

User:Andy Maloney/Surface passivation effects on kinesin and microtubules

Andy Maloney — Wed, 28 Jul 2010 14:14:54 GMT

Notebook:

←Older revision		Revision as of 14:14, 28 July 2010
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	\|addcategories=true		\|addcategories=true
	\|category=Surface passivation		\|category=Surface passivation
-	~~\|uses=Template:AndyMaloneyNotebook~~
-	~~\|includepage={AndyMaloneyNotebook}:[[%PAGE%]]~~
-	~~\|includepage={AndyMaloneyNotebook}:Description~~
-	~~\|format=,\n[[%PAGE%\|%TITLE%]]\n'''Categories:''' %CATLIST%\n*'''Description:''' ,,~~
-	}}
-	~~{{#dpl:~~
-	~~\|namespace=User~~
-	~~\|order=descending~~
-	~~\|addcategories=true~~
-	~~\|category=AM_SurfacePassivationMTs&Kinesin~~
	\|uses=Template:AndyMaloneyNotebook		\|uses=Template:AndyMaloneyNotebook
	\|includepage={AndyMaloneyNotebook}:[[%PAGE%]]		\|includepage={AndyMaloneyNotebook}:[[%PAGE%]]

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

Andy Maloney — Wed, 28 Jul 2010 14:13:05 GMT

Entry title:

New page

User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/27/Reboot

Andy Maloney — Wed, 28 Jul 2010 14:12:29 GMT

←Older revision		Revision as of 14:12, 28 July 2010
Line 1:		Line 1:
	{{AndyMaloneyNotebook		{{AndyMaloneyNotebook
-	\|Description=	+	\|Description=Initial test to see if all my new solutions work. No data.
	}}		}}
-	[[Category:Surface ~~Passivation~~]]	+	[[Category:Surface passivation]]
	==New solutions==		==New solutions==
	After remaking all of my expired solutions, I am running an experiment now. I'm using whole casein as the surface passivator since it is my baseline for data. Everything seems to be working fine and I'll just have to wait till the computer crunches the numbers before I can say if everything is working as I think it should.		After remaking all of my expired solutions, I am running an experiment now. I'm using whole casein as the surface passivator since it is my baseline for data. Everything seems to be working fine and I'll just have to wait till the computer crunches the numbers before I can say if everything is working as I think it should.