Biology Reference
In-Depth Information
Atomic Force Microscopy (AFM)-based methods can apply large indentation stresses
(
0.1-10 kPa) to soft interfaces and provide simultaneous topographical information,
but cannot easily probe the three-dimensional properties of polymer solutions or
gels (
Kirmizis & Logothetidis, 2010
). Optical trapping methods provide nanometer
resolution of probe position and can operate at high frequencies. However, optically
transparent materials of fairly low index of refraction are required, and since the max-
imum applied force is typically tens of pico-Newtons, use of optical trapping methods
has been limited to fairly soft materials (
50 Pa) (
Preece et al., 2011; Valentine et al.,
2008
). For both optical traps and AFM, the application of constant force requires
computer-controlled feedback to compensate for instrument compliance.
Magnetic tweezers devices provide a valuable alternative, allowing for charac-
terization of three-dimensional materials while providing
<
nano-Newton forces.
However, previous implementations of magnetic tweezers for microrheology have
typically relied on the use of electromagnets operating at high current, which can
heat samples and exhibit a hysteretic response, or on extremely small distances
(
microns) between the pole pieces and magnetic beads, or both (
Bausch,
M
¨
ller, & Sackmann, 1999; de Vries, Krenn, van Driel, & Kanger, 2005;
Kollmannsberger & Fabry, 2007; O'Brien, Cribb, Marshburn, Taylor Ii, &
Superfine, 2008; Spero et al., 2008
). These constraints can lead to unusual experi-
mental geometries with potential interference in high-resolution microscopy, can
create steep force gradients within the image plane, and, in the case of devices in
which iron pole pieces are submerged into the sample, can create chemically reactive
metal ions. By contrast, neodymium iron boron (NdFeB)-based magnetic tweezers
are noninvasive and easily provide constant force to the sample plane without the use
of feedback control. Because of these advantages, NdFeB magnets have become a
standard technology for single-molecule force spectroscopy where femto- to pico-
Newton forces are required; however, these have thus far found limited utility in
meso- to macroscale materials characterization, which typically requires larger
forces to achieve measurable deformations. In this chapter, we describe the design
and construction of three newmicroscope-mounted NdFeB-based magnetic tweezers
devices optimized for use with MT networks. Our protocols outline all aspects of
instrument assembly and calibration, MT network preparation, structure determina-
tion, as well as mechanical data collection and analysis. With these devices, we have
successfully characterized many aspects of MT mechanics.
6.1
PROTOCOLS
6.1.1
Preparation of samples
6.1.1.1
Preparation of tubulin proteins
The protocols for unlabeled and rhodamine-labeled tubulin proteins have been pre-
viously described in detail. Briefly, unlabeled tubulin is purified from bovine brain
by cycles of assembly and disassembly and followed by phosphocellulose
