Biology Reference
In-Depth Information
through, using a Kim-wipe type tissue to wick out fluid from the opposite side of the
flow lane. A particularly important side note: be sure no bubbles flow through the
lanes; the surface-bound proteins must always remain in solution. We build the glid-
ing assay by first coating the surface with biotinylated BSA, blocking the surface
with BSA, adding SA to create a biotin-SA layer, removing excess SA, adding bio-
tinylated kinesin to form a bio-SA-bio-kinesin layer, washing out free kinesin, and
washing in microtubules in the appropriate solution. The steps to this protocol are as
follows:
1.
Construct flow cell with
4 lanes.
2.
Wash bio-BSA into each lane (10-15
m
l/wash depending on size of lane).
3.
Wash lane 3
with BSA (
15
m
l/wash).
4.
Wash SA into lane.
5.
Wash 3
with BSA.
6.
Wash 1
with CBAB.
7.
Dilute kinesin 1:100 in CBAB, wash
15
m
l/lane, wait no less than 5 min
(10 min seems optimal, but up to 2 hours works), and make FAB during this wait
time.
8.
Wash 1
1/100 paclitaxel (40
m
M paclitaxel).
9.
Dilute microtubules 1:100-1:1000 in FAB
with CBAB
þ
þ
ATP (
1 mMworks well), wash in,
and observe.
We observe the fluorescently labeled microtubules using a home-made TIRF micro-
scope (
Friedman, Chung, & Gelles, 2006
); however, any microscope capable of im-
aging single fluorophores with approximately 20 nm precision at 200 ms will work.
At 1 mM ATP, kinesin propels microtubules at approximately 0.5
m
m/s, depending
on the temperature of the experiment. We observe microtubules in a 50
50-
m
m field
of view, so an individual microtubule is observable for approximately 100 s. Using
an EM-CCD camera (Andor iXon DV-897), we image the microtubules at 5 Hz,
choosing an illumination intensity that does not photobleach the fluorophores over
the 100 s of observation. The typical fluorophore intensity we observe is approxi-
mately 1000 photons per fluorophore per 200-ms image. See
Fig. 2.3
A for a typical
image of microtubules in the gliding assay.
2.2.3
Image analysis and persistence length calculation
The first step in the analysis is extracting the trajectories of individual fluorophores
from the sequence of 500 images (100 s, 5 Hz) collected in the
Section 2.2
. We use a
modified version of software developed by
Crocker and Grier (1996)
for this anal-
ysis, which results in trajectories for each fluorophore (i.e., an ordered set of posi-
tions with time for each position). As an aside, an excellent web tutorial on
particle tracking, useful for exactly this analysis, is maintained by
Weeks (2013)
.
If this resource is accessible, it provides an ideal gateway into single fluorophore
tracking.
