User:Andy Maloney/Kinesin & Microtubule Page: Difference between revisions

Revision as of 11:00, 9 March 2011

Purpose

This page describes the various procedures and solutions I use for gliding motility experiments. Some insight and information into the chemicals and tools used to run these experiments are given. As well as failures I have encountered.

This is the first chapter in my completely open notebook science dissertation. If you would like to post questions, comments, or concerns, please join the wiki and post comments to the talk page. If you do not want to join the wiki and would still like to comment, feel free to email me.

Introduction



Figure 1: Abstract cartoon of a kinesin and microtubule protofilament. The blue circles are the cargo binding groups, the red is the stalk, the brown is the neck linker and the yellow are the motor domains. The microtubule protofilament is made up of alpha (pink) and beta (green) tubulin.

All experiments require a certain number of tools necessary in order to make them work. These tools may be chemicals and proteins, equipment, or even procedures. It's easy to call chemicals and proteins tools since they are nothing more than the ingredients for an experiment, much like flour and eggs are ingredients for a cake. Equipment of course works well with this analogy since there is no way one can make the cake without an oven. Similarly, you will never be able to observe an experiment without equipment to do so. Procedures are somewhat different and may or may not fall into my analogy so easily. However, my definition of a tool is something used without the need for creativity. Using my definition, procedures are then nothing more than the directions to make an experiment work. Or, to use my analogy of a cake, procedures are nothing more than the recipe to make the cake. Now, coming up with the procedure may require creativity and the use of equipment in novel manners but, once the recipe has been perfected, it becomes a tool. I can extend this analogy even further by saying that the final product, a cake, is nothing more than a canvas for creativity. As popular TV chefs of this era (who decorate cakes) will understand⁽¹⁾, the cake can be a tool or a vehicle for their creative arts. Not to belittle the mastery of baking, because it is tough and not everyone can follow a recipe properly. The preparation of an experiment is the tool for a scientist to be creative about science.

It is thus extraordinarily important for a scientist to be able to prepare an experiment with the utmost precision possible. That way, the prepared tools are the same consistently. If the tools are produced the same all the time, this allows the scientist the leisure of not having to worry about an outcome changing simply because the time of day changes or other black box scenarios. The same thing applies to the baker, without the consistency of the cake recipe and ingredients, customers are not guaranteed a tasty cake on Wednesday as opposed to Thursday. Since, as we know, Wednesday is the day that we don't silence other people's cannons⁽²⁾.

For gliding motility assays, I use a great number of tools. These tools come in the form of chemicals and proteins, equipment, and procedures. While some of the information in this page may seem way over bearing and incredibly "nit picky", I have found that by understanding how and why I use everything for a specific purpose helps me to troubleshoot problems that may arise in the assay due to some fault of a tool. As with any biological experiment, there are numerous variables and any number of those variables can cause problems in an experiment. By understanding the tools used, I can rule out problems in the final product easily and, I can hunt down problems and fix them when they do creep up.

Along with the tools used for making a gliding motility assay, I will also discuss some of the failures I have come across when making these assays. Thankfully I have been able to collect enough data to show how this assay can go awry and, I hope that can be of some use to others who do these types of experiments.

Chemicals, solutions, suspensions, proteins, and flow cells

There are a lot of chemicals used in gliding motility experiments. The fun part of these experiments are when you can change the chemicals to try and fish out some physics between the interactions of kinesin and microtubules. Before I talk about changing chemicals, I will describe my procedure on how to make the basic buffer solution used for nearly all experiments; the PEM buffer. I will then go into how to make the other buffers and solutions necessary to run the most basic gliding motility assay.

The PEM buffer

As stated above, it is extremely important to get this right and to be able to make this buffer consistently the same every time. PEM stands for:

And for pH-ing

NaOH

I have adopted this terminology since it is the easiest for me to remember and, it goes well with the naming of the other solutions necessary for the experiment. Links are to the exact product I use for the buffer. Before talking about the procedure for making the buffer, I will digress and discuss a little bit about each chemical.

PIPES is an acid and should be treated as such. It is the pH buffer chemical in the solution and it has a pK_a of 6.76. The lab chose to use the acid form due to the literature. In the literature, there is no consensus to which form of PIPES to use and if either sodium or potassium is better to use as the counter ion for the gliding motility assay. See for instance Ray et.al.⁽³⁾ for an example of a buffer pH-ed with KOH and Woehlke et. al.⁽⁴⁾ for an example using NaOH. We decided to use the acid form of PIPES so that we can actively choose which counter ion we want in solution, be it from NaOH or KOH. So, my PEM buffer should probably be called Na-PEM since I use NaOH to pH my buffer and to get PIPES into solution. If I were to use KOH, then I would probably call it K-PEM. PIPES should be stored at room temperature in the desiccator in its original bottle.

EGTA chelates both calcium and magnesium from solution and is also an acid. Thankfully, EGTA has a higher affinity for calcium than it does for magnesium since for motility to work, you need magnesium in solution⁽⁵⁾. I have yet to find out why we use EGTA in the PEM buffer especially since we use very pure chemicals and 18.2 MΩ-cm water. Perhaps it is a legacy chemical used in initial experiments because people wanted to get rid of any trace amounts of calcium in solution. I believe that we still use EGTA in PEM is because EGTA will chelate calcium phosphate found in casein micelles. The chelation of calcium phosphate will break apart the micelles and possibly might aid in surface passivation⁽⁶⁾. Fox and McSweeney state that EDTA will disintegrate casein micelles but they do not talk about EGTA disintegrating casein micelles. Holt et. al.⁽⁷⁾ does talk about the similarities of how EDTA and EGTA was used to investigate the ratio of calcium to phosphate in milk. This makes me inclined to believe that EGTA will disintegrate casein micelles like EDTA does. In fact, I believe that it may do a better job of breaking up the micelles since EGTA has a higher affinity for calcium than EDTA does. Our acid form of EGTA will not go into solution without cations in the form of NaOH or KOH also in solution. Again, we chose the acid form of EGTA for the same reasons why we chose the acid form of PIPES. EGTA should be stored in the desiccator at room temperature in its original bottle.

Magnesium is essential for both the polymerization of microtubules⁽⁵⁾ and for the motility of kinesin⁽⁸⁾. This is why magnesium chloride is included in the PEM buffer. I purchase MgCl₂ in solution at a concentration of 1 M. I do not purchase the salt form of MgCl₂ because it is extraordinarily hygroscopic. So much so that if you leave a pellet of it out, it will suck up so much water from the atmosphere that it will basically put itself into solution. Even in the New Mexico dry air. Since MgCl₂ is in solution already, it does not need to be stored in the desiccator and it can be stored at room temperature in its original bottle.

As I mentioned above, we use the salt forms of PIPES and EGTA. Both require that there be counter ions present in solution in order for them to become soluble. We have chosen to use sodium in the form of NaOH as our counter ion which will also pH the solution. Getting the correct amount of NaOH for a solution of PEM was tricky at first, however, now I know the approximate amount to use from trial and error. Scientists typically will not state how much of a pH-ing solution is added to a buffer. I'm not sure why this is because changing the ionic strength of the motility assay does affect it⁽⁹⁾. NaOH comes in pellet form and should always be dessicated due to its hygroscopic nature. You must work with this chemical quickly when weighing it out, otherwise it will pull water from the atmosphere and throw off your measurement. NaOH should be stored at room temperature in the desiccator at all times.

The PEM recipe & procedure

My PEM buffer is not unique but, neither is it the standard. See here for a few other labs' PEM buffers. In fact, there is no standard buffer for running gliding motility assays and more to the point, there is no standard to the naming convention for this buffer. PEM can also be called BRB80 and as the link will suggest, no one really knows where this terminology (BRB80) came from.

The final recipe I have decided that works well for the experiments I run is the following:

800 mM PIPES
10 mM EGTA
10 mM MgCl₂
[math]\displaystyle{ \approx }[/math] 1.25 M NaOH

It is a 10x concentrated solution that is diluted to 1x before experiments are conducted. The procedure I use to make this buffer is the following:

I weigh out the appropriate amount of PIPES, EGTA, and NaOH to make a total volume of 25 mL. The exact amount of 1.25 M NaOH is not necessary. In fact, since it is an approximate concentration value, I will weigh out slightly less NaOH than the 1.25 M concentration. I typically keep it around 1.22 - 1.24 M NaOH. That way I don't end up having to pH the buffer with HCl and if the solution needs it, I can add more NaOH if needed. I place all these components in a 50 mL centrifuge tube and vortex them together with a small amount of water (typically 10-15 mL of water) to get all the chemicals in solution. I also add the MgCl₂ in this step.
Once all the chemicals are in solution, I add more 18.2 MΩ-cm H₂O to the tube till the total volume is about 22 mL. I don't add the total amount of volume needed (25 mL) since I know that I may have to pH the buffer. Having less than the total volume needed ensures that I don't add too much water to the solution thus diluting the chemicals.
I next determine the pH of the solution and add either NaOH or HCl to the solution. In all honesty, if I have to add HCl to the solution I typically scrap the whole lot. This is because I do not want excess ions floating around in my experiments. I finally make a note of how much NaOH I needed to properly pH the buffer.
I add the appropriate amount of water to reach the 25 mL total volume mark.
I then syringe filter the buffer using a 0.2 µm filter and aliquot into 1 mL aliquots and store in screw top vials that are then labeled and stored in the 4˚C fridge in a convenient fridge box.

The antifade system

I use a very common antifade system that consists of the following parts.

Catylase (CAT)
Glucose Oxidase (GOD).
[math]\displaystyle{ \beta }[/math]-mercaptoethanol (BME).
2 M D-glucose in PEM (PEM-Glu).

The antifade system is integral for a good gliding motility assay. There are several reasons why I use it and the number one reason why it is used is that it prolongs the observation time of fluorescent microtubules. Since I observe the microtubules with fluorescence, elongating the time it takes before the microtubule fades is crucial for taking good data with a high signal to noise ratio. I rather dislike using this antifade system and there are other recipes for antifade systems that exist⁽¹⁰⁾, however, I have not preformed any experiments with the PCD antifade systems.

Glucose oxidase (GOD) requires D-glucose in solution as this is its fuel source. GOD oxidizes D-glucose to gluconic acid while using up oxygen in the solution. This is a good thing since photobleaching is caused from highly reactive oxygen species. Unfortunately when GOD oxidizes D-glucose, it also produces hydrogen peroxide. Catalase (CAT) is added to the mix in order to decompose the hydrogen peroxide. BME, [math]\displaystyle{ \beta }[/math]-mercaptoethanol, or 2-mercaptoethanol is used to prevent blinking of the fluorophore and to quench triplet states^(10,11).

Along with the above two solutions, I make up what I call PEM-Glu. PEM-Glu is nothing more than a 2M solution of D-glucose in PEM. Yes. I want to make a 2 M solution of glucose, which means that there is a lot of sugar to put into a very small volume of liquid. This is an extreme case where I have to really take into consideration the volume of the solute that I'm putting into solution. If I measure out the total volume of liquid I want to make a 2 M solution of glucose with and just add the glucose to it, I will end up with a solution that is a larger volume than I anticipated. To prevent this from happening, I weigh out the glucose and add it to a smaller volume of liquid than my final target total volume. I vortex it so that a considerable portion of the glucose goes into solution and then add more PEM till I reach my target volume. I only make 1 mL total volume of PEM-Glu and aliquot it into 20 µL aliquots and store them in the -80˚C freezer. D-glucose should be stored at room temperature in its original container. I should note that the PEM-Glu is not added to the antifade system aliquots, it's added to the motility solution before observations.

The final component of the antifade system is the BME. BME is nasty stuff and smells quite terrible. If any of this stuff spills anywhere, the stench will permeate through the lab for days. Even if it's just a microliter it will stink up the place very badly. Anything that touches BME needs to be handled carefully and disposed of, or cleaned properly.

The antifade system recipe and procedure

Antifade systems are made with 100x GOD and 100x CAT solutions in PEM. The appropriate concentrations for the 100x stock solutions are below.

2000 µg/mL Glucose oxidase in PEM
800 µg/mL Catalase in PEM
20% (v/v) BME

Weighing out glucose oxidase and catalase for these solutions at 100x is tough due to the small amount needed to be weighed out. I opt for making a 1000x concentrated solution of the chemicals and then dilute them by a factor of 10 to obtain the 100x solutions needed for the cocktail. Both the glucose oxidase and the catalase should be stored in their original containers in the -20˚C fridge. I have them in a secondary container with desiccant.

The recipe and procedure for this antifade system is as follows.

I add 12 µL of 100x GOD to a microcentrifuge tube.
I add 12 µL of 100x CAT to the same microcentrifuge tube.
In the hood with the fan on, I add 6 µL of BME.
I vortex the solution and spin it down so that I can aliquot it into 5 µL aliquots. I and store the cocktail the -20˚C freezer.

I should note that the antifade system should not freeze while in the freezer. If it does, then something has gone wrong with the preparations and the aliquots should not be used. I've also found out that the cocktails only keep for approximately one to two weeks in the -20˚C freezer.

Tubulin suspension in TSB

The tubulin I use comes from bovine brains and is purchased from Cytoskeleton. Thankfully there is a company in which I can purchase tubulin from since I'm not too keen on having to harvest tubulin myself which involves liquefying brains⁽¹²⁾. I have three different types of tubulin in the lab:

The tubulin is lyophilized (flash frozen) and comes from Cytoskeleton in 1 mg, 20 µg, and 20 µg aliquots respectively. Aliquots are stored in the -80˚C freezer at all times upon arrival. My gliding motility studies use rhodamine labeled tubulin exclusively, as it gives a much better signal to noise ratio than does the fluorescein labeled tubulin.

To prepare the tubulin for easy polymerization, I store it in what I call TSB or Tubulin Storage Buffer. The limited number of studies I have looked at show that tubulin polymerizes into microtubules best in PEM⁽⁶⁾. I believe this is why we use PEM in everything and TSB is no exception as TSB is just PEM with extra stuff in it. I use the following recipe to prepare TSB.

1.06x PEM
1 mM MgCl₂. This is an extra 1 mM above what is already in the PEM.
1 mM GTP.
6% (v/v) Glycerol

The extra 1 mM MgCl₂ is added since microtubules will not polymerize effectively without it in solution⁽¹²⁾. The lab has chosen to add an extra 1 mM in the TSB since EGTA chelates Mg²⁺ from solution and I'd much rather have too much MgCl₂ in solution than not enough.

Figure 2: Image of GTP bottle and its insert.

GTP comes packaged nicely in 10 mg jars so I do not have to weigh it out. It is highly toxic so I use caution when opening the jar. Preparing this suspension in the hood is highly recommended in order to prevent any inadvertent inhalation of GTP dust, as it definitely burns a lot when inhaled. I add the 1.06x PEM to the GTP bottle such that the GTP is at 100 mM. In order to get all the GTP from the bottle into solution, I have to slosh the contents around after adding the PEM. The GTP bottle has an insert in it that I pull out for easier mixing.

The reason as to why I put 100 mM GTP in a 1.06x concentrated solution of PEM and not the 1x PEM is because the GTP will be used exclusively in the TSB. TSB gets diluted by 6% (v/v) when adding the glycerol hence the need for GTP to be in a higher concentration of PEM. I transfer the GTP + 1.06x PEM mix into a screw top vial and flash freeze it with LN₂. I store it in the -80˚C freezer. I have used both a diluted PEM solution and one that takes into consideration the dilution of 6% when adding the glycerol. Both work fine in polymerizing microtubules.

Glycerol is extraordinarily viscous and is a terrible pain to try and measure out. The way I measure it out is with the 1000 µL pipettor. The 1000 µL pipet tip is large enough such that I can get the glycerol to go into it and I've found that glycerol won't go up a 100 µL pipet tip at all. Getting all the glycerol needed in the pipettor takes some zen motivation as it takes patience to wait till all the glycerol is in the tip. As Heinz ketchup would say back in the 1980's "Good things come to those who wait."

Glycerol is used to speed up microtubule polymerization⁽¹³⁾. Other chemicals can be used in polymerization from DMSO to excess Taxol. These three polymerization techniques result in three different types of microtubules being polymerized with the major difference in the microtubules being the number of protofilaments⁽³⁾.

GTP suspension

To prepare the GTP in 1.06x PEM I follow the below recipe.

1.06x PEM:
- 2820 μL 18.2 MΩ-cm H₂O.
- 180 μL 10x PEM

GTP suspension:
- 191.14 μL 1.06x PEM.
- Add to the GTP bottle and mix. Place in a screw top vial and store in the -80°C freezer.

TSB recipe and procedure

I prepare a 2 mL solutions of TSB. I do this mainly so that I am in the lower range of the 1000 mL pipettor for glycerol measurements. If stored properly in the -80˚C freezer, TSB will last up to a year. Unfortunately, there is no way anyone will use all the 2 mL TSB solution before it goes bad. My recipe is below.

1858 μL 1.06x PEM
120 μL Glycerol
20 μL 100 mM GTP in 1.06x PEM
2 μL MgCl₂

Tubulin dimers are highly unstable, so care should be taken to keep them as cold as possible and to minimize steps that keep the tubulin from being frozen. Once I have the TSB prepared, I am ready to prepare aliquots of tubulin that can be used for polymerization into microtubules.

Un-labeled tubulin suspension

Un-labeled tubulin comes packed in vials containing 1 mg of tubulin. I suspend this tubulin to a final concentration of 5 mg/mL in TSB in convenient aliquots.

I remove a vial of tubulin from the -80˚C freezer and put it in the e•IceBucket to defrost. If necessary, I spin the vial to get all the tubulin to settle at the bottom. Be careful though since tubulin is very labile and may be destroyed during this step.
I then suspend the tubulin in 200 µL of TSB. I mix the solution by gently drawing the tubulin + TSB mixture back into the pipettor and blow it out again into the vial. I will do this three time.
I then aliquot into 5 µL aliquots in the 200 μL microcentrifuge tubes, flash freeze in LN₂ and store in the -80˚C freezer.

Labeled tubulin suspension

Rhodamine labeled tubulin and fluorescein labeled tubulin come packed in vials containing 20 µg of tubulin. I will suspend either of these tubulins to a final concentration of 5 mg/mL.

I remove a vial of tubulin from the -80˚C freezer and put it in the e•IceBucket to defrost. If necessary, I spin the vial to get all the tubulin to settle at the bottom. Be careful though since tubulin is very labile and may be destroyed during this step.
I then suspend the tubulin in 4 µL of TSB. I mix the solution by gently drawing the tubulin + TSB mixture back into the pipettor and blow it out again into the vial. I will do this three time.
I then aliquot into 2 µL aliquots in the 200 μL microcentrifuge tubes, flash freeze in LN₂ and store in the -80˚C freezer.

29% Labeled tubulin suspension

Using 100% rhodamine labeled tubulin in an experiment is not ideal. I have found an acceptable ratio of labeled tubulin to unlabeled tubulin using the 100W Hg lamp with 6% illumination. 29% rhodamine labeled tubulin to 61% unlabeled tubulin gives a great signal to noise ratio and the microtubules can be tracked easily. Since both my unlabeled and labeled tubulin are now in convenient aliquots, I will use them to make up a 29% rhodamine labeled suspension. I do this by:

I will dethaw one aliquot of the rhodamine labeled tubulin and unlabeled tubulin each in the electronic ice bucket.
I add the unlabeled tubulin to the labeled tubulin.
I mix gently with the pipettor to ensure that the two solutions are mixed properly.
I then aliquot into 1 μL aliquots and store in the -80°C freezer in the 500 μL microcentrifuge tubes. I will typically use a 25 mL centrifuge tube to store my tubulin aliquots in.

These 29% labeled tubulin suspension are what I will use in experiments. See below for a description of how I polymerize the tubulin into microtubules.

Taxol suspension

Taxol is purchased from Cytoskeleton and is stored in the -80˚C freezer in a container filled with desiccant. Taxol is used to stabilize the microtubules as they are quite labile and will break apart easily⁽¹⁴⁾. Breaking apart easily is a spectacular characteristic of microtubules as they are essentially roads, for motor proteins, under constant construction and deconstruction in the cell. The cell can determine if a road is no longer needed or if it needs to be repaired or elongated thus allowing the motors that travel along them to deliver items to different parts of the cell. This "instability" is essential for cell function and in mitosis. A good example of this instability is shown in the below video.

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Movie 1: Dynamic instabilities of microtubules.

The above movie is from the Yamada Lab at UC Davis. This video shows that the ends of microtubules, labeled with GFP, grow and shrink dynamically. While this is good for cells, it is not good for gliding motility experiments as I am measuring speeds at which the microtubules float across a sea of kinesin. If the microtubule under observation suddenly shrinks, this will not allow me to get a good measure of its speed. To prevent the breakup of microtubules (depolymerization) I use Taxol.

Taxol is an anti-cancer drug⁽¹⁵⁾ that stabilizes microtubules. Since cancer cells are fast growing cells, Taxol helps to slow down the spread of the tumor by inhibiting microtubule dynamics and thus cellular replication. The stabilizing effect of Taxol is a good thing for my experiments and is why it is used.

Taxol is hydrophobic so it will not go into an aqueous environment unless the solution has DMSO in it. DMSO is an amazing chemical that solubilizes Taxol such that I can add it to a PEM solution. DMSO is hygroscopic and reacts with just about everything. An interesting side note about DMSO is that if it gets on your skin, you will taste garlic almost immediately. Sigma packs DMSO in ampules and the liquid needs to be transferred into a different screw top container for easier access. Since DMSO likes just about everything, it must be stored in something that it will not react with. DMSO can be stored in HDPE, LDPE, and PP without any problems. The DMSO from one ampule can be stored in 3 cryo vials. These cryo vials are then stored in a secondary HDPE container filled with desiccant in a nitrogen environment and stored in the desiccator.

10 mM stock Taxol suspension procedure

The Taxol I get from Cytoskeleton has approximately 170 μg of Taxol in the vials. Adding 20 μL of DMSO will make a 10 mM Taxol in DMSO solution.

One vial of Taxol from Cytoskeleton.
20 μL of DMSO.

I vortex the vial and spin it down for use. I always store the 10 mM Taxol solution in the e•IceBucket when not in use since it is set to 3˚C. DMSO freezes at 3˚C so this is a nice way to see if the solution is still good or not. If the solution does not freeze, this means that the DMSO has absorbed enough water from the atmosphere to render it unreliable for experiments.

If there is too much Taxol in solution or, if the Taxol stock has gone bad, I will get Taxol spindles as depicted in the movie I took below. For an explanation on how Taxol forms these spindles see^(16-18).

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Movie 2: Taxol crystals found in one of my assays. Note that the crystal does not photobleach.

If this occurs, I make a new stock solution of Taxol. The worst case scenario when this does happen is that the pipettor has gone out of calibration and I have put too much Taxol in solution. Thankfully, that doesn't happen too often and Taxol crystals typically only occur for me when my Taxol solution has stopped freezing at 3˚C.

There are some things to take note of about this spindle. The first thing is that the fibers are rigid. If I observe what I think is a microtubule but it doesn't bend at all, then it is more than likely a Taxol crystal. Also, the spindle does not photobleach very much. The above movie was observing the spindle with no ND filters in front of the Hg lamp. If this was a bunch of microtubules and not a Taxol crystal, the microtubules would have depolymerized quickly leaving nothing to observe.

PEM-A

ATP is the fuel for kinesin. It is what kinesin uses in its motor domains to produce a step on a microtubule. To prepare PEM-A, I use:

PEM
ATP

Since I have a salt form of ATP, it goes into solution very easily. ATP should be stored in a secondary container that is filled with desiccant and under a nitrogen environment in the -80˚C freezer.

The book, Molecular Cloning⁽²¹⁾, says to suspend ATP in a Tris buffer at pH 8.0 since ATP auto-hydrolyzes less in alkaline buffers as opposed to acidic ones. Alberty⁽²²⁾ shows a graph that the auto-hydrolysis of ATP doesn't change very much for ATP stored in buffers at pH 7. Since PEM is pH-ed to 6.89, I figured it would be just fine to store the ATP in it. I have not had any problems using ATP stored in PEM and I believe that not having to introduce another chemical, namely the Tris, into the assay is beneficial. I will note that after storage, the PEM-A solution will start to look cloudy. I will mix the solution as best as I can and spin the precipitate to the bottom of the tube using the mini centrifuge. I'll decant the fluid such that I do not get any of the precipitate in my assays.

Recipe and procedure

100 mM ATP in PEM
I'll aliquot into 10 μL vials and store them in the -80°C freezer.

Flow cell

The above link shows an old method to how I prepared my flow cells. Rather than get rid of it entirely, I have opted to include the link so that it is easier to show the evolution of the flow cell preparation. I put some notes by the video pointing out where I did things incorrectly.

Below, you will find my updated version of how I create my flow cells.

Surface passivation chemicals

In order to sustain motility, the flow cell must be passivated. This is commonly done with bovine whole casein. I have done studies using all components of bovine casein and have determined that the best surface passivation protein to use is alpha casein ⁽¹⁹⁾. Bovine serum albumin is another protein commonly used as a surface passivator^(5,9).

Bovine milk has four major proteins in it; α_s1, α_s2, β, and κ casein. Each mammal species has a different mixture of casein constituents in their milk. Different species' milk is evolutionarily tuned for their neonates and differ considerably between species. For instance, human milk contains β, κ, and only trace amounts of α_s1 caseins as opposed to the four constituents that bovine milk has⁽⁶⁾. Another fact about milk is that each mammal in a species will produce milk that has a distinct amount of caseins in it. While the species as a whole will have on average the same percentage of casein in their milk, cow A may have 35% β casein in it's milk (the average) while cow B may have 37% of β casein in its milk.

This variability is not really a problem in the lab due to the fact that the milk we use undoubtedly came from a farm that stuck hundreds of cow's milk in a giant vat. That giant vat would then be sent to the purifiers to make the casein we purchase. This averaging over many cows is a good thing for an experimenter but, the variability is something to take into consideration.

Below, you will find links to my recipes for preparation of the various bovine casein solutions that can be used for gliding motility assays.

W-PEM
- 1.0 mg/mL of whole casein in PEM.
[math]\displaystyle{ \alpha }[/math]-PEM
- 1.0 mg/mL of α casein in PEM.
[math]\displaystyle{ \beta }[/math]-PEM
- 1.0 mg/mL of β casein in PEM.
[math]\displaystyle{ \kappa }[/math]-PEM
1.0 mg/mL of κ casein in PEM.
M-PEM
- This solution has the appropriate mixture of α-PEM, β-PEM, and κ-PEM in it such that the amount of proteins equal roughly those in the average cow's milk⁽⁶⁾.

Microscope and software

In this section, I will describe the microscope that I use and briefly introduce the software that has been designed in Dr. Koch's lab, mainly by Larry Herskowitz for the observation, tracking, and automated data analysis of the gliding motility assays.

Programs and equipment

I use an Olympus IX71 inverted microscope setup for epifluorescence using a 100W Hg lamp from Olympus. The objective is an Olympus PlanApo 60x 1.42NA. The filter cube is a TRITC filter cube with Chroma filters in it. The microscope also has ultraviolet neutral density filters and I typically will illuminate my microtubules with 6% of the 100W Hg lamp. I have made a cover for my microscope out of blackout material and PVC pipe. The cover is really cheap and it blocks out stray light as well as keeping dust and strong air currents from affecting measurements. My objective has extra stuff attached to it that I will talk more about below.
I use an Andor Luca-S camera with custom LabVIEW acquisition software written by Larry Herskowitz. The acquisition software captures .png files from the camera (grey background program) and are time stamped and stored for later analysis. Another custom LabVIEW program written by Larry is used to convert the series of .pngs captured by the camera to .avis (pink background program). The camera is attached to a 3 axis stage as well as a rotation stage and not directly attached to the microscope. This allows me to center the camera in the field of view easily and reduces noise in the acquisition due to the camera's fan.
After the images are captured, another custom LabVIEW software written by Larry is used to automatically track microtubules as they glide over kinesin in the assays. This program searches a parent directory of pngs and tracks the minus and plus ends of the microtubules through pattern matching algorithms.
Once the microtubules have been tracked, yet another custom LabVIEW program (again, thanks to Larry) is used to analyze the data. This program loads data from the tracked microtubules and performs data smoothing and kernel density estimation analysis on the speeds of individual microtubules. It can do so in an automated manner and has the capability of analyzing thousands of microtubule tracks relatively quickly.
The current computer used to capture and analyze data has the following specs.

Objective heater

Temperature stabilization is crucial for obtaining stable speed measurements. I will talk more about why in a following chapter. I have followed the work done by Mahamdeh and Schaeffer⁽²⁰⁾ pretty closely with my objective heater build.

Materials

Most of the materials can be purchased from TeTech. The rest can be purchased from Mouser.

Build

The temperature controller I purchased came in its own aluminum box with exposed screw terminals. I didn't like the idea of having exposed wiring from the control box so I made a break away box with banana connectors on it. It isn't pretty but, it gets the job done. I also connected an RS232 connector to the box in order to communicate with the computer. There are several connectors going to this box. 2 for power, 2 for power to the heater, and 4 connectors for 2 different thermistors. The control thermistor and the sensor thermistor.
I taped the larger thermistor to the base of the objective and placed the flexible heating element above it. The heating element comes with glue on it so all I did was stick it to the objective. There are 2 alligator clips attached to the heating element that supply current to it from the control box. The thermistor on top is epoxied into place with Arctic Silver Thermal Epoxy.
The power supply is an OEM power supply and so it has exposed terminals on it. I enclosed the power supply in a box and chose to have a main switch to power the power supply (green indicator LED) and another DPST switch on the front to turn on the +12VDC output to the temperature controller. This allows the power supply to warm up before switching power to the temperature controller. To the right is a picture of the circuit in the box.
If I could have found a bigger switch on the front, then I would have used it. Why? Because everybody loves switches.

Experiments

Once I have all my stock solutions prepared, I am now in a position to start putting them all together. There is still some preparatory work that needs to be done before an experiment can be observed in the microscope however.

Assay checklist

Before starting an experiment, I always ensure that I have the following solutions and stocks prepared ahead of time.

10x PEM
H₂O - just a convenient vial of water is necessary to dilute the 10x PEM.
PEM
29% labeled tubulin
Antifade
10 mM Taxol in DMSO
PEM-Glu
α-PEM
PEM-A
PEM-T
PEM-α
PEM-αA
Kinesin

I have not spoken about PEM-T, PEM-α, PEM-αA, or kinesin yet and before I outline my recipe to make the motility assay, I will talk about these solutions.

PEM-T is what I use to fix microtubules that have been recently polymerized. The recipe for this is:

10 μM Taxol in PEM.

This Taxol solution is in an aqueous environment. This means that I never keep a stock solution of it lying around and I will make a fresh batch of it every time I polymerize microtubules. I will make up a 199 μL solution of PEM-T by mixing up the following.

198.8 μL PEM
0.2 μL of the 10 mM Taxol in DMSO.

I make this solution about 5 minutes before the microtubules are supposed to come out of the thermal cycler. I do this to help ensure that there will not be a large number of Taxol crystals in solution and that most of the Taxol will be used to stabilize my fresh microtubules.

PEM-α is just a dilution of α-PEM. Instead of 1.0 mg/mL of alpha casein in PEM, it is 0.5 mg/mL alpha casein in PEM. PEM-α is used to dilute the kinesin in and I make up

500 μL α-PEM
500 μL PEM

1 mL typically.

PEM-αA is PEM-α plus 1 mM PEM-A. PEM-αA is what is used to store the kinesin that will be used in an assay in. I will make up the following:

99 μL PEM-α
1 μL PEM-A.

Kinesin was supplied by Dr. Haiqing Liu at a concentration of 275 μg/mL should be stored in the -80°C freezer at all times. When I'm ready to conduct experiments, I will take an aliquot of kinesin out of the freezer and place it in the electronic ice bucket. Once the kinesin has been removed from the freezer, I never put it back as in doing so will cause the kinesin to deteriorate faster than if it stays in the electronic ice bucket at 4°C.

Once all the solutions I need are ready, I'll start an experiment.

Making an assay and microtubule polymerization

The first step I take is to turn on the mercury lamp and setup the microscope for Kohler illumination. I will then make sure that the camera software is ready to take data. The mercury lamp should be on for at least 30 minutes before taking measurements to ensure that it is warmed up and ready for experiments.
Once the microscope is setup, I'll make sure that the temperature controller is on and the software associated to it is working properly.
While I'm setting up the microscope, I'll ensure that the thermalcycler is on and warming up for polymerization. Once it is, I will take an aliquot of tubulin from the freezer and place it in the thermalcylcer. The tubulin should be in the thermalcycler for a total of 30 minutes.
Around 20-25 minutes I will prepare the solution of PEM-T.
Once the 30 minutes are up, I will add the PEM-T to the microtubules while in the thermalcycler. I will then remove the tube and protect it from ambient light and store it at room temperature.

Once I have microtubules, I am now ready to start the preparation of a slide for an assay.

I add 10 μL of the α-PEM to the flow cell and allow it to sit for 10 minutes.
Before the 10 minutes are up, I will dilute 1 μL of kinesin in 9 μL of PEM-αA and store it in the electronic ice bucket. This diluted solution of kinesin is at a concentration of 27.5 μg/mL.
Still during the 10 minutes, I will prepare a motility solution. Motility solutions consist of
- 90.5 μL PEM
- 1 μL PEM-Glu
- 1 μL PEM-A
- 2.5 μL Antifade
- 0.1 μL Taxol
- 5 μL of microtubules
Once the 10 minutes are up, I will add the diluted kinesin in PEM-αA to the flow cell by fluid exchange. This then is allowed to sit for another 5 minutes.
After the 5 minutes are up, I add 10 μL of the motility solution to the flow cell by fluid exchange.
I seal the flow cell with nail polish and put it on the microscope to observe the system.

Conclusion

The gliding motility assay is fickle to put it politely. It is full of possible issues that can cause the completed flow cell to not exhibit motility. This is the worst case scenario as it takes anywhere from 1 to 2 hours to produce the first flow cell of the day. Thankfully making another one only take any where from 15-30 minutes. The above recipe works well for me as it has allowed me to create checks with chemical aliquots that help prevent failure of an experiment.

References

Ace of Cakes
The Adventures of Baron Munchausen paraphrase
Ray, S., Meyhöfer, E., Milligan, R., & Howard, J. (1993). Kinesin follows the microtubule's protofilament axis. The Journal of Cell Biology, 121(5), 1083-1093. doi: 10.1083/jcb.121.5.1083.
Woehlke, G., Ruby, a K., Hart, C. L., Ly, B., Hom-Booher, N., & Vale, R. D. (1997). Microtubule interaction site of the kinesin motor. Cell, 90(2), 207-16.
Böhm, K. J., Steinmetzer, P., Daniel, A., Baum, M., Vater, W., & Unger, E. (1997). Kinesin-driven microtubule motility in the presence of alkaline-earth metal ions: indication for a calcium ion-dependent motility. Cell motility and the cytoskeleton, 37(3), 226-31. doi: 10.1002/(SICI)1097-0169(1997)37:3<226::AID-CM4>3.0.CO;2-4.
Fox, P., & McSweeney, P. (1998). Chapter 4: Milk Proteins. Dairy chemistry and biochemistry (1st ed., pp. 146-238). London: Blackie Academic & Professional.
Holt, C., Ormrod, I. H., & Thomas, P. C. (1994). Inorganic constituents of milk. V. Ion activity product for calcium phosphate in diffusates prepared from goats’ milk. The Journal of dairy research, 61(3), 423-6. doi: 10.1017/S0022029900030855.
Olmsted, J. B., & Borisy, G. G. (1975). Ionic and nucleotide requirements for microtubule polymerization in vitro. Biochemistry, 14(13), 2996-3005. doi: 10.1021/bi00684a032.
Böhm, K. J., Stracke, R., & Unger, E. (2000). Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters. Cell biology international, 24(6), 335-41. doi: 10.1006/cbir.1999.0515.
Aitken, C. E., Marshall, R. A., & Puglisi, J. D. (2008). An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophysical journal, 94(5), 1826-35. doi: 10.1529/biophysj.107.117689.
Rasnik, I., McKinney, S. a, & Ha, T. (2006). Nonblinking and long-lasting single-molecule fluorescence imaging. Nature methods, 3(11), 891-3. doi: 10.1038/nmeth934.
Shelanski, M. L., Gaskin, F., & Cantor, C. R. (1973). Microtubule assembly in the absence of added nucleotides. Proceedings of the National Academy of Sciences of the United States of America, 70(3), 765-8.
Keates, R. A. B. (1980). Effects of Glycerol on Microtubule Polymerization Kinetics. Biochemical and Biophysical Research Communications, 97(3), 1163-1169. doi: 10.1016/0006-291X(80)91497-7.
Arnal, I., & Wade, R. H. (1995). How does taxol stabilize microtubules?. Current biology, 5(8), 900-8. doi: 10.1016/S0960-9822(95)00180-1.
Yvon, a M., Wadsworth, P., & Jordan, M. a. (1999). Taxol suppresses dynamics of individual microtubules in living human tumor cells. Molecular biology of the cell, 10(4), 947-59.
Foss, M., Wilcox, B. W. L., Alsop, G. B., & Zhang, D. (2008). Taxol crystals can masquerade as stabilized microtubules. PloS one, 3(1), e1476. doi: 10.1371/journal.pone.0001476.
Castro, J. S., Trzaskowski, B., Deymier, P. a, Bucay, J., Adamowicz, L., & Hoying, J. B. (2009). Binding affinity of fluorochromes and fluorescent proteins to Taxol™ crystals. Materials Science and Engineering: C, 29(5), 1609-1615. Elsevier B.V. doi: 10.1016/j.msec.2008.12.026.
Castro, J. S., Deymier, P. a, Trzaskowski, B., & Bucay, J. (2010). Heterogeneous and homogeneous nucleation of Taxol crystals in aqueous solutions and gels: effect of tubulin proteins. Colloids and surfaces. B, Biointerfaces, 76(1), 199-206. doi: 10.1016/j.colsurfb.2009.10.033.
Passivation paper place holder.
Mahamdeh, M., & Schäffer, E. (2009). Optical tweezers with millikelvin precision of temperature-controlled objectives and base-pair resolution. Optics Express, 17(19), 17190. doi: 10.1364/OE.17.017190.
Sambrook, J., Russell, D.W. (2001). Molecular cloning : a laboratory manual (3rd ed.). Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory Press.
Alberty, R. A. (1968). Effect of pH and Metal Ion Concentration on the Equilibrium Hydrolysis of Adenosine Triphosphate to Adenosine Diphosphate. The Journal of Biological Chemistry, 243(7), 1337-1343.

Quick reference materials list

Below is a quick reference list of the items used in this assay.

Chemicals

Equipment

Supplies & Tools

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