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chromatography ( Miller &Wilson, 2010 ). Rhodamine-labeled tubulin is prepared by
reaction with succinimidyl esters of carboxyrhodamine-6G (C-6157; Invitrogen)
( Hyman et al., 1991 ). We find that using a molar ratio of rhodamine-labeled tubulin
to total tubulin of 1:6 is sufficient for visualization using a point-scanning confocal
microscope (Olympus Fluoview 500). In some cases, we desire to assess the effects
of chemical cross-linking on MT structure and mechanics. For these experiments,
we include some fraction of commercial biotinylated porcine brain tubulin
(T333P; Cytoskeleton, Inc.) that has been labeled at a
1:1 ratio of biotin to tubulin
heterodimer and free streptavidin (SA20; Prozyme, Inc.). The biotinylated tubulin is
delivered as a lyophilate and reconstituted to 10 mg/mL in G-PEM80 buffer (80 mM
PIPES, 4 mM MgCl 2 , 1 mM EGTA, and 1 mM GTP; pH 6.9).
6.1.1.2 Preparation of MT networks embedded with magnetic beads
Entangled MT networks are formed by combining the following reagents on ice:
unlabeled and rhodamine-labeled tubulin, 1 mM DTT, 10% (v/v) DMSO, and taxol
in G-PEM80 buffer. We typically include taxol (semisynthetic, T7191; Sigma-
Aldrich) at a molar ratio of 1:2 taxol to total tubulin to prevent dynamic length
changes during the course of the measurement, and we perform the majority of
our measurements at room temperature. The typical tubulin concentration ranges
from 10 to 50 m M in our measurements. If the effects of dynamic growth and shrink-
age on network properties are of interest, then taxol may be omitted; however, in its
absence, the polymerized MT solution must be maintained at
35 C by a heating
stage and/or objective warmer to prevent MT disassembly. The DMSO promotes MT
polymerization, and the DTT minimizes disulfide bond formation and photodamage.
Tosyl-activated magnetic beads with diameter of 4.5
m (Dynabeads; Invitrogen)
are added to the ice-cold mixture at a final concentration of
m
10 6 beads/mL. In some
cases, we seek to visualize the long-range deformation field induced by the motion of
the magnetic bead and include 2.5-
m
m latex beads (PS05N; Bangs Laboratories Inc.)
10 7 beads/mL as fiducial markers.
The ice-cold tubulin solution is then loaded into small rectangular tubes
(0.1
at a final concentration of
5
50 mm 3 ; Friedrich & Dimmock, Inc.) by capillary action; the resultant
sample volume is
1
L. Prior to loading, the capillary tubes are cleaned by rinsing
with 1 M sodium hydroxide, then precoated with reference beads to enable the sub-
traction of artifactual mechanical or thermal drift, or vibration of the sample, and/or
the stage from the real motion of the embedded magnetic particles. To achieve this,
5
m
5.4
m
m latex beads (PS06N; Bangs Laboratories, Inc.) are diluted in isopropanol to
10 5 beads/mL and loaded into the capillary tubes. The tubes are placed on a flat
benchtop for 10 min to promote sedimentation. The tubes are gently dried and baked
at 150 C for 2 min to partially melt the beads onto one side then allowed to cool
completely before immediate use.
After introduction to the capillary tubes, entangled networks are immediately
sealed with high-vacuum grease and placed in a dry incubator at 35 C. It is critical
that this process proceed quickly to avoid formation of structures that are shear aligned
by loading and to avoid sedimentation of the magnetic beads. The filled capillary tubes
5
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