Effects of Surface Passivation on Gliding Motility Assays
Andy Maloney*, Lawrence J. Herskowitz, Steven J. Koch
Department of Physics and Astronomy and Center for High Technology Materials, University of New Mexico, Albuquerque, New Mexico, United States of America
Abstract
In this study, we report differences in the observed gliding speed of microtubules dependent on the choice of bovine casein
used as a surface passivator. We observed differences in both speed and support of microtubules in each of the assays.
Whole casein, comprised of as1, as2, b, and k casein, supported motility and averaged speeds of 96667 nm/s. Alpha casein
can be purchased as a combination of as1 and as2 and supported gliding motility and average speeds of 94964 nm/s. Beta
casein did not support motility very well and averaged speeds of 870630 nm/s. Kappa casein supported motility very
poorly and we were unable to obtain an average speed. Finally, we observed that mixing alpha, beta, and kappa casein with
the proportions found in bovine whole casein supported motility and averaged speeds of 96666 nm/s.
Citation: Maloney A, Herskowitz LJ, Koch SJ (2011) Effects of Surface Passivation on Gliding Motility Assays. PLoS ONE 6(6): e19522. doi:10.1371/
journal.pone.0019522
Editor: Laurent Kreplak, Dalhousie University, Canada
Received November 18, 2010; Accepted April 6, 2011; Published June 3, 2011
Copyright:  2011 Maloney et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The experiments were funded by DTRA grant number ‘‘HDTRA-1-09-1-0018’’ and the INCBN from a NSF Grant ‘‘DGE-0549500’’. The funders had no role
in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: amaloney@unm.edu
Introduction
Kinesin-1 (hereafter referred to as kinesin) is an ATPase that
converts chemical energy to mechanical work. It travels along
microtubules in one direction and can carry with it various cellular
items [1–5]. In vitro motility studies use two different methods to
investigate the kinesin and microtubule system. In one method,
microtubules are fixed to a coverglass and individual kinesin
motion are observed either by single-fluorophore tracking, or by
attaching beads to kinesin [6–8]. The other method is a gliding
motility assay where kinesins are fixed to a glass slide and
microtubules flow on top of a layer of kinesin [9–11]. In the gliding
motility assay, motility is sustained by first passivating the glass to
prevent kinesin’s motor domains from becoming inactive when
interacting with untreated glass.
Passivation of glass can be done with bovine serum albumin
(BSA) [9–11], bovine casein [12–16], a lot of kinesin [17], or other
compositions [18]. Bovine casein is the typical surface blocker
used, mainly because it works well at passivation and is
inexpensive. Casein is a globular protein that does not have a
known crystal structure [17]. Bovine casein is comprised of four
major subgroups: as1, as2, b, and k. Depending on the mammal
the caseins come from, there exists different ratios of these globular
constituents. For instance, bovine casein contains as1+as2.b.k
and human casein contains b.k with only trace amounts of as1
casein [19,20].
How casein passivates a glass surface in order to support kinesin
for the gliding motility assay is still not very well understood.
However, some work has been done to try and elucidate how
casein passivates glass surfaces. Ozeki et al. showed that two layers
of casein form on the glass surface to help support kinesin for
motility [12]. Verma et al. [17] also investigated how kinesin and
casein interact depending on which casein constituent from bovine
milk was used. In their study, they showed that the number of
microtubules that landed on the kinesin surface was affected by the
casein passivation. Hancock and Howard also showed that the
number of microtubules that landed on the kinesin surface was
dependent on the number of motor proteins adhered to the glass
slide [21]. Building on these prior studies, we investigated whether
the gliding speed of microtubules was affected by the type of casein
used to passivate the glass slide.
Materials and Methods
Open data and open notebook science
Raw data and all open notebook entries regarding this
experiment are publicly available [22].
Microscopy and software
Experiments were conducted on an Olympus IX71 inverted
microscope using an Olympus 6061.42 NA PlanApo objective.
Rhodamine fluorophores attached to tubulin were illuminated
with a 100 W mercury lamp (attached to the microscope and
attenuated by 94%) using a TRITC filter cube with Chroma’s
filter set 49005. The strong attenuation was to help reduce
photobleaching and potential local heating of the sample. Image
sequences were captured using custom LabVIEW software with an
Andor Luca S camera. Data analysis was done with custom
LabVIEW tracking software. A description of the tracking and
camera software will follow in another paper, however, a link to
the readme file for the software can be found here [22].
The microscope objective was held at a constant temperature
using a polyimide film resistive heater, a temperature controller
from TeTech, two 15 kV thermistors, and LabVIEW software.
The design was very similar to the work done by Mahamdeh and
Scha¨ffer [23] and a complete description of our design can be
found in the supporting information Text S1. Briefly, the objective
was thermally isolated from the objective turret using a spacer.
The control thermistor was attached to the objective near its base
and the control circuit from TeTech monitored it and used the
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heater, placed directly above the thermistor, to heat the objective.
The other thermistor was bonded to the top of the objective using
thermal epoxy and was used as the temperature probe. This design
can maintain a temperature to 60.1uC. There are very few studies
that indicate whether or not observation of the gliding motility
assay was done with temperature stabilization or not. It has been
shown that temperature does play a crucial role in obtaining stable
data [10,11] and we have also seen the effects of non temperature
stabilization in our own data, see the supporting information Text
S2. Due to the thermal connection with the mercury arc lamp, the
entire microscope body heats up with a time constant of several
hours. Without temperature stabilization, this caused a steady
increase in gliding speed over the course of several hours [24].
Flow cells
Gliding motility assays were performed using custom flow cells.
A detailed description for the construction of the flow cells can be
found in the supporting information Text S3. Briefly, a slide and
cover slip were sandwiched together using double stick tape. The
tape was positioned on the slide such that a channel of
approximately 10 mL volume was formed. After trimming excess
tape, a cover slip was placed over the channel and pressed to
ensure proper adhesion to the tape. Once the flow cell had been
prepared for observation, nail polish was used to seal the open
ends. We have not observed any adverse affects to the assay by
using nail polish as the sealant nor have we tried other brands than
the one we use regularly, which is made by NYC.
Buffers and solutions
The preferred solution to conduct gliding motility assays in is
usually called BRB80 [25], and is also known as PEM. We prepare
a 10x PEM solution containing: 800 mM PIPES (Sigma 80635),
10 mM EGTA (Sigma 80635), 10 mM MgCl2 (Sigma M1028)
and pH-ed to 6.89 using approximately 1.25 M NaOH (Fisher
S318) in 18.2 MV-cm water. The amount of NaOH was
approximate since each solution of PEM was pH-ed to 6.89.
Bo¨hm [11] showed that gliding speed was affected by both the pH
and the ionic strength of the solution the motors were in. In an
effort to reduce as many variables as possible for speed
measurements, we chose to maintain the pH of our PEM buffers
at exactly 6.89 and vary the amount of NaOH necessary to
achieve this pH. All buffers and solutions were prepared in
18.2 MV-cm water produced with a Barnstead EasyPure RoDI
system. The 10x PEM stock solution was diluted by a factor of 10
at the time of experiments. The PIPES and EGTA we used were
acid forms of the chemicals to allow us the ability to choose
different cations to add to the solution in the form of NaOH or
KOH. Once pH-ed, the PEM solution was passed through a
0.2 mm syringe filter and aliquoted in 1 mL screw top vials and
stored at 4uC.
A mixture of the as1- and as2-caseins purified to 70%, b-casein
purified to 98%, and k-casein purified to 70% were purchased
from Sigma (C6780, C6905, and C0406 respectively). Each casein
component was dissolved in PEM under constant stirring. a-casein
took approximately 60–80 minutes of constant stirring before no
more precipitate was visible in solution, b-casein took approxi-
mately 30–40 minutes, and k-casein required 15–20 minutes, all at
room temperature. Visible whole casein (Sigma C7078) precipitate
remained in solution if no heat was applied. Whole casein is not
susceptible to thermal denaturation and was not affected by
moderate heating [26]. Heating PEM to 60u–80uC while stirring
in whole casein, caused the visible precipitate to be dissolved in
PEM. We used a condenser to prevent evaporation of the buffer
while heating. See the supporting information Text S4 for a
detailed description for adding whole casein to PEM. All casein
solutions were reconstituted to 1.0 mg/mL in PEM. After all the
casein was dissolved in solution, it was stored at 4uC in convenient
aliquots with no additional filtering. Casein solubility is a
complicated function of other casein constituents in solution
[27,28], temperature [29–31], genetic variants [32], ionic strength
and types of salts in solution [32], calcium ion concentration
[27,28,32], and pH [33]. Casein solubility measurements were not
performed in this study.
Microtubules were polymerized from bovine tubulin purchased
from Cytoskeleton. We used both unlabeled (TL238) and
rhodamine-labeled bovine tubulin (TL331M), stored as lyophilized
aliquots at 280uC. Tubulin was polymerized in PEM, with the
addition of 1 mM GTP (Sigma G8877), an extra 1 mM MgCl2,
and 6% (v/v) glycerol (EMD GX0185). The inclusion of an extra
1 mM MgCl2 was to ensure that the EGTA in PEM did not
chelate all the magnesium ions from solution since tubulin
polymerization requires magnesium ions [25]. Adding glycerol to
the storage/polymerization solution speeds up microtubule
polymerization [34]. Polymerization was carried out in a Thermo
PCR Sprint thermocycler held constant at 37uC and incubated for
30 minutes using 29% rhodamine-labeled and 71% unlabeled
tubulin. After 30 minutes, the microtubules were fixed and diluted
by 200x with a solution of PEM with 10 mM Taxol (Cytoskeleton
TXD01) and removed from the thermal cycler. Our typical
polymerization volume was 1 mL. After the polymerization cycle
was complete, we added 199 mL of our PEM plus Taxol solution
to stabilize the microtubules. Taxol is not highly-soluble in water
and must be suspended in DMSO (Sigma D2650). We
reconstituted Taxol to a final concentration of 10 mM in DMSO
and stored it in a Bionexus eNIceBucket at 3uC. Taxol can create
crystals in aqueous solutions due to its low solubility. It also has a
high affinity for free tubulin and or rhodamine dye molecules
[35,36]. This can cause Taxol crystals to appear to be fluorescent
microtubules [37]. In order to reduce the prevalence of Taxol
crystals, any solution containing Taxol was always prepared fresh
and immediately before experiments. No stock solutions contain-
ing Taxol in an aqueous environment were prepared for future
use. Polymerized microtubules were stored at room temperature
and protected from ambient light until used in a motility assay.
Storing polymerized microtubules at 4uC will cause rapid
depolymerization [34].
Our kinesin was generously supplied by Dr. Haiqing Liu and
was supplied to us in 20 mL aliquots at a concentration of
0.275 mg/mL kinesin. The kinesin is his-tagged, truncated
kinesin-1 dmk401 [38,39]; from drosophila and was expressed in
E. coli. Kinesin was diluted to 27.5 mg/mL for each assay.
Motility assays
Motility assays contained 10 mM Taxol, 1 mM Mg-ATP (Sigma
A9187), 20 mM D-glucose (Sigma 49139), 2.5% (v/v) of an
oxygen scavenging antifade system, and 5 mL of fixed polymerized
microtubules for a total volume of 100 mL in PEM with no added
casein. The kinesin home page [40], as well as Verma et. al. [17],
suggest that the inclusion of casein to the motility solution will
enhance the chances of a gliding motility assay to work properly.
We have observed that adding casein to the motility solution
caused microtubules to undergo non ideal motility, i.e. they moved
in tight circles and ended up wrapping around themselves such
that the microtubules looked like squiggles and were untrackable.
In order to prevent such behavior, we do not include casein in our
motility solution. Our antifade system was a dual enzymatic
oxygen scavenging system that contained in the stock solution;
800 mg/mL glucose oxidase (Sigma G6641), 2000 mg/mL catalase
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(Sigma C9322) and 20% (v/v) of 2-mercaptoethanol, referred to as
BME, (Sigma 63689). When diluted into the motility solution,
there was 8 mg/mL glucose oxidase, 20 mg/mL catalyase and
0.5% (v/v) BME. Antifade cocktails were prepared in advance and
stored in 5 mL aliquots at 220uC. We have noticed that the
antifade system will remain viable at 220uC for only one week.
Beyond one week of storage, the antifade solutions were disposed
of and fresh aliquots were prepared when needed.
Flow cells were constructed and incubated at room temperature
(usually between 24uC and 25uC) with the various caseins for 10
minutes. Kinesin was then diluted in PEM with 1 mM Mg-ATP
and 0.5 mg/mL of the same casein used for the passivation in a
ratio of 1:10 kinesin:buffer for a final kinesin concentration of
27.5 mg/mL of kinesin in solution. The kinesin was introduced
into the flow cell by fluid exchange and allowed to incubate for
another 5 minutes. Finally, our motility solution was flowed into
the cell that was then sealed with nail polish to prevent
evaporation.
Experiment and data collection
After sealing the flow cell, the slide was immediately placed on
the microscope. Data was taken at 5 frames per second with each
frame having an exposure of 100 ms and a EMCCD gain of 150.
Multiple regions on the slide were exposed and images were
collected for at least 20 regions of interest (ROIs) for each slide.
Each ROI was exposed for approximately 2 minutes allowing the
camera and computer to collect 600 total frames.
Data was then analyzed using customized LabVIEW (National
Instruments, Austin, TX) automated microtubule tracking soft-
ware. A detailed description of the tracking software will follow in
another paper [41]. Briefly, microtubules were identified by NI
Vision 7.1 image segmentation algorithms and the ends of
microtubules were indentified via pattern matching algorithms.
Tracking of a microtubule was stopped if it got too close to the
edge of the field of view or, if it overlapped with another
microtubule. Data were discarded for microtubules tracked for
fewer than 100 consecutive image frames. Tracks were also
discarded for microtubules with a segmented area less than 55
pixels. This filtering prevented tracking of microtubules that were
either too small for the image recognition to precisely locate the
ends of the microtubule or microtubule tracks that did not have
enough data points for the subsequent speed analysis.
Automated tracking provided the x and y position with subpixel
accuracy of the microtubule ends for each frame that was tracked.
These time series data were smoothed with a sliding Gaussian
window with a standard deviation of 2 seconds. Smoothed data
points that were within 5 seconds of the beginning or end of the
tracks were discarded in order to eliminate boundary effects on the
smoothed data due to the window. Smoothing the position versus
time data was necessary since the microtubule ends are never
permanently attached to the kinesin surface and thus undergo
transient Brownian motion. After smoothing and truncating, the
instantaneous speed was calculated as the distance per time
between consecutive frames.
Speed versus time data for all the microtubules in an individual
ROI were then concatenated together and the most likely speed
was extracted using a kernel density estimation (KDE) [42] with a
Gaussian kernel of width 50 nm/s. The ROIs were 2 minutes
long, had 600 images, and could have one to over 100
microtubules that needed to be tracked. We used a KDE method
instead of a simple mean because we wanted to reduce our
sensitivity to microtubule pausing or stalling, which was evident in
many assays. We used a large kernel width to reduce sensitivity to
possible speed changes due to number of kinesin motors or other
causes [43]. The most likely speeds for individual regions were
then plotted versus time to determine when the slide had reached
thermal equilibrium with the objective. The initial 5 data points
were removed for all data sets indicating that it took about 10
minutes for the slide to reach a stable temperature. Each assay;
alpha, beta, kappa, whole, and mixed casein was repeated 3
separate times on different days and with different kinesin aliquots.
When possible, the mean and standard error of the mean was
computed for the three data points for a given assay time. Time
differences of 620 seconds were ignored for this calculation.
Results and Discussion
Results
Data was taken at 33.160.1uC as measured from the top
thermistor on the objective. This temperature is well above the
temperature the objective would reach due to long-term heating
from the Hg lamp and was found to give consistent data. We
observed (data not shown) that the closer the observation was to
the boundaries of the flow cell, the slower the microtubule gliding
speed was. We also observed that the propensity for depolymer-
ization increased near the boundaries. In order to obtain
consistent data and prevent depolymerization of the microtubules,
gliding assays were observed in the center of the flow cell channel,
except where otherwise noted. All images have had dead pixels
removed by an interpolation function and have been false colored
using ImageJ’s green fire blue LUT using a custom LabVIEW 7.1
application.
Bovine alpha casein (a mixture of as1 37% and as2 10%)
constitutes approximately 47% of whole bovine casein [20]. This
passivation was capable of supporting small microtubules and
longer ones as can be seen in Figure 1A. When using alpha casein,
the gliding motility assay worked every time except when we
deemed the kinesin or antifade system to have lost its effectiveness
for maintaining a gliding assay.
Bovine beta casein comprises approximately 35% of whole
casein and visible precipitates took less time to dissolve in PEM as
compared to alpha casein. Figure 1B shows that beta casein as a
surface passivator was not ideal. It did not support smaller
microtubules and did not in general have very many motile
microtubules in any assay. The microtubules that were motile in
this assay were typically quite long. Beta casein also caused the
microtubule’s minus and positive ends to detach from the kinesin
surface while undergoing motility more often than the other
passivation schemes. This caused errors in the tracking and thus
did not give consistent data. However, it can be purified to better
than 98% making this component of whole casein the purest
commercially available. Another interesting phenomenon ob-
served when using beta casein was that motile microtubules were
not found in the center of the channel for the flow cell. Motile
microtubules were observed, but they were always found off center
to the flow cell channel. We do not know the reason for this, and it
may depend on kinesin or casein concentration. We did not vary
the input kinesin or casein concentration for these studies.
Kappa casein, compared to alpha and beta, is structurally very
different. It is a glycoprotein and is thought to stabilize the casein
micelle [20,44,45] by sterically hindering the aggregation of too
many casein sub-micelles. It did not support motility in a very
consistent manner as can be seen from Figure 1C&D. As was the
case for beta casein, kappa casein did not support motility in the
center of the channel of the flow cell. Stuck microtubules were
found near the center of the flow cell and near the tape. However,
between the boundaries of the flow cells and away from the center
of it, there was motility. In the areas that motility existed, kappa
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casein was able to support motility of long and short microtubules
with the exception of extremely short microtubules found to move
only in the alpha or whole casein assays. The very short
microtubules either remained stuck to the surface or exhibited
motility for a very short period of time before going into solution.
We also observed that kappa casein did a remarkable job of
adhering microtubules to the slide much like how poly-L-lysine is
used to fix microtubules to glass [46].
Whole bovine casein is the passivator of choice when doing
gliding motility experiments. We found that whole bovine casein
worked remarkably well for sustaining motility. Similar to the
alpha casein passivation, whole casein worked every single time
and gave consistent data. It was only when we deemed either the
kinesin or the antifade system to have lost its effectiveness at
maintaining microtubule gliding that the assay did not work. Of
the 5 types of bovine casein solutions tested, whole casein required
heat in order for visible precipitate to completely dissolve in PEM.
See the supporting information Text S4 for a more thorough
discussion of how we dissolve whole casein in PEM. We did not
observe any adverse affects to motility by heating whole casein.
Whole casein supported long and short microtubule motility as
shown in Figure 1E.
Mixing whole casein from the individual constituents of alpha,
beta, and kappa was also used as a surface passivator. The mixed
whole casein consisted of 49% alpha casein, 37% beta casein and
14% kappa casein which was very similar to what Fox and
McSweeney state as the casein components of bovine milk [20].
Mixing it was easy since each component was already in a PEM
solution. The behavior of the mixed bovine whole casein was
indistinguishable from the purchased whole casein or the alpha
casein passivation. The number of microtubules and the varying
lengths undergoing motility in the mixed casein passivation was
very similar to those of the whole casein passivation as well.
Figure 2 shows a histogram of microtubule lengths. To obtain
lengths, we used an erosion algorithm on binary images of only the
microtubules that were tracked. We used a standard function in
LabVIEW/Vision 7.1 called Skeleton L. After the erosion, we
used a Convex Hull perimeter calculation also found in the
LabVIEW/Vision 7.1 library of functions. We divided the Convex
Hull perimeter by 2 to estimate the microtubule length. Two of the
authors independently and manually estimated the length of
several microtubules and found that usually the Convex Hull
perimeter was in between both manually obtained values. We
decided that this was sufficient for identifying large changes in MT
length distributions, and so we did not further investigate the
robustness or systematic errors in our length estimation. As can be
seen in the figure, alpha casein (filled blue bars), whole casein
(empty purple line bars) and mixed casein (empty black line bars)
all were able to sustain motility with smaller microtubules 2–6 mm
range. Beta casein (filled green bars) had significantly fewer
microtubules that were tracked, however, those that were tended
to be longer than the ones tracked in the alpha casein assay.
Kappa casein (filled red bars) show that there were many more
trackable microtubules than beta casein and significantly fewer
than alpha casein. The smallest trackable microtubules using our
algorithm are not smaller than 2 mm. Figure 2 shows that while in
comparison to the other lengths, there are relatively few 2 mm
microtubules in any of the assays, kappa casein had no
microtubules that fell in the 2–3 mm range. There is, however, a
larger number of kappa casein microtubules that were in the 20–
21 mm range as compared to the other assays. Length measure-
ments using this method are not optimized for precision, but this
Figure 1. Observations of microtubules in flow cells passivated by different types of casein. Images have been false colored with
ImageJ’s Green Fire Blue LUT and have had dead pixels removed using an interpolation function. A. Alpha casein (Sigma C6780) passivation showed
support for both long and short microtubules. B. Beta casein (Sigma C6095) passivation did not support motility very well and did not support
shorter microtubules. C. Kappa casein (Sigma C0406) did support motility but only in limited regions of the flow cell. In other regions near the center
of the channel (D) the microtubules were stuck to the slide and did not exhibit motility. E. Commercially available whole casein (Sigma C7078) is the
standard for surface passivation for the gliding motility assay. It supported both long and very short microtubules. F. Mixed casein which was made
from 49% a, 37% b, and 14% k casein worked just as well as alpha casein and whole casein passivation in terms of number and sizes of microtubules
exhibiting motility.
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method does give a simple way to see the relative size distribution
differences in the assays.
Figure 3 shows the mean speed measurements for 15 different
regions of interest for the alpha, beta, whole and mixed casein
assays. Each data point is the mean of a region of interest with
SEM from three separate samples. The passivator that gave the
most consistent speed was alpha casein. The mean speed and SEM
from our alpha casein measurement was 94964 nm/s. Purchased
whole casein and mixed casein performed remarkably similarly
and displayed average speed values of 96667 nm/s and
96667 nm/s respectively. Bovine beta casein performed poorly
in comparison to alpha, whole, or mixed caseins and we measured
the mean speed to be 870630 nm/s. Figure 4 shows the observed
speeds for kappa casein passivation. Since there were so many
areas where no motility was observed in this assay, it was difficult
to determine a mean speed measurement for each assay as was
done in Figure 3. However, it does appear that when motile, the
speeds were around 870–880 nm/s with kappa casein as the
surface passivator. This was similar to how beta casein performed.
Discussion
We observed that the more difficult it was to dissolve casein, the
better it worked as a surface passivator. This may be a
coincidence, or it may relate to the manner in which casein
adsorbs to the glass. Whole casein was by far the most difficult to
dissolve visible precipitate into PEM without heating. Alpha casein
came in second, beta third and kappa fourth in terms of the time
to dissolve completely in PEM at room temperature. Whole casein
is approximately 50% alpha casein and seeing how well alpha
casein performed as a surface passivator, we were not surprised
that whole casein also performed well. The differences in
measured speeds between alpha and whole casein could be a
result of how kinesin was supported by the different caseins, or it
could be due to differences in surface-coverage by the casein. It
has been shown that using glass of differing hydrophilicity with
whole casein passivation affects the activity of kinesin [47]. In this
study, we observed a difference in speed due to the type of casein
passivation.
Purchased whole and mixed whole casein had indistinguishable
speeds, and no other observable differences. Mixed whole casein
has an upper bound of 20% impurities in it. This amount was very
similar to the amount of impurities alpha casein has in it yet,
mixed whole casein performed exactly how purchased whole
casein did. This similarity between mixed and purchased whole
casein suggests that the speed difference between alpha and whole
casein was not due to impurities but rather that it was due to how
kinesin was supported by the casein micelles or, how casein
interacts with the glass. To elucidate the effects, it would be
prudent to measure the speeds for the various caseins as a function
of casein and kinesin concentrations during incubation. We have
not yet performed these experiments.
Beta casein would have been the most ideal protein to use as the
surface passivator since one can purchase it to greater than 98%
purity. Higher protein purity is advantageous for systematically
producing devices that use kinesin and microtubules as sensors.
However, beta casein did not perform very well. It had the least
number of motile microtubules to track, it was not very reliable,
and had a large distribution in speeds. It is possible, though, that
varying other parameters such as kinesin concentration, or beta
casein concentration could restore reliable motility. We have not
yet attempted these experiments.
Kappa casein would be an attractive surface passivator just for
its ease in dissolving in PEM. However, where motility exists in the
Figure 2. Relative microtubule length distributions. Filled blue
bars are length calculations for the alpha casein passivation, filled green
bars are for the beta casein passivation, filled red bars are for the kappa
casein passivation, unfilled black bars are for the mixed casein
passivation, and unfilled purple bars are for the whole casein
passivation. Length measurements were performed only on tracked
microtubules and are estimations from computing the Convex Hull
perimeter on eroded images of microtubules.
doi:10.1371/journal.pone.0019522.g002
Figure 3. Speed versus assay time for four types of casein
passivation. Black circles are alpha casein passivation, red squares are
beta casein passivation, green up pointing triangles are whole casein
passivation, and blue down pointing triangles are mixed casein
passivation. Each data point is the mean from three different samples,
taken at approximately the same assay time. Error bars represent the
standard error of the mean. Alpha casein had the most consistent
average speed measurements at 94964 nm/s. Whole casein and mixed
casein averaged to 96667 nm/s and 96666 nm/s respectively. Beta
casein averaged to 870630 nm/s.
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flow cell was not consistent and it never occurs in the center of the
channel where we have already observed the most consistent speed
measurements from other assays. With kappa casein, we observed
many long and stable microtubules permanently stuck to the
surface. We are not sure if the sticking was caused from kinesin
attaching to microtubules and then somehow being impeded from
moving or, if there was actually no kinesin on the kappa casein
surface and the microtubules are just attracted to the kappa casein
or the glass.
Of the five types of bovine casein used to observe the gliding
motility assay, alpha casein performed very well. It was the easiest
of the three commercially available bovine casein constituents to
dissolve in PEM and can be purchased at 70% purity. Most likely,
the 30% contaminants are from other casein components. It
performed well every time an assay was prepared and worked just
as well as mixed and purchased whole casein.
Conclusion
There are a wide variety of surface passivation strategies in the
literature. Even for use of kinesin in gliding motility assays, where
casein is the most commonly used passivator, there are many
varieties to choose from. We found that alpha casein (Sigma
C6780) was the most reliable passivator when following the most
typical gliding assay protocol. We are currently using this as our
passivator for studies on the effects of water isotope and osmotic
stress on kinesin microtubule activity. We did not explore the
parameter space of kinesin or passivator concentration or
incubation temperature or time. Statistically designed experiments
Figure 4. Gliding speed measurements with kappa casein passivation. Green squares, red circles, and blue triangles represent 3 different
assays. As the figure shows, kappa casein was not the ideal surface passivator. There were many regions in the flow cell with no motility and other
regions that showed inconsistent motility and at much slower speeds than was reported from alpha, whole or mixed casein passivations. The open
black circles show a characteristic alpha casein assay for comparison. Note that the initial increase in speed (time less than 500 seconds) was due to
the slide coming to thermal equilibrium with the objective. Because the three kappa casein samples appeared to have different speed distributions
and because many regions of interest showed no motility, we did not attempt to compute a mean speed versus assay time, as was done in Figure 3
for the other caseins.
doi:10.1371/journal.pone.0019522.g004
Casein Effects on Gliding Motility Assays
PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e19522

(DOE) may be an efficient method for optimizing the assay
conditions for other casein varieties.
Supporting Information
Text S1 Objective heater. Temperature stabilization of the
objective was done with an objective heater. The following text
describes the build to the objective heater.
(DOC)
Text S2 Temperature data. A description and graph
showing the importance of temperature stabilization of the
objective.
(DOC)
Text S3 Flow cell construction. Here we describe in detail
how we made the flow cells used for this study.
(DOC)
Text S4 Whole casein in PEM. Here we describe in detail
how we dissolved whole casein in PEM.
(DOC)
Acknowledgments
We would like to thank Dr. Haiqing Liu for supplying kinesin to us. We
would also like to thank Dr. Erik Scha¨ffer for his detailed discussions about
temperature stabilization and Dr. Susan Atlas for technical discussions.
Author Contributions
Conceived and designed the experiments: AM SJK. Performed the
experiments: AM. Analyzed the data: AM LJH SJK. Contributed
reagents/materials/analysis tools: AM LJH SJK. Wrote the paper: AM
LJH SJK. Designed the software used in the analysis: LJH SJK.
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