Biology Reference
In-Depth Information
13.1.2.1.2.4
Protofilament defect microtubules.
Microtubules polymerized with
GTP and stabilized with Taxol display 12-13 protofilaments (
Arnal & Wade,
1995
), while microtubules polymerized with GMPCPP contain mostly 14 protofila-
ments (
Hyman, 1995
). In order to create microtubules with defects, we end-to-end
anneal these two types of microtubules together after each has formed. This occurs
spontaneously at high concentration, as evidenced by striped microtubules in solu-
tions of older polarity-marked microtubules.
1.
Create GMPCPP and Taxol-stabilized microtubules as described above. If using
the same fluorophore on both, we recommend a 10-fold difference in labeling
concentrations for the two types.
2.
Shear each set of microtubules by passing the microtubules through a Hamilton
syringe (gauge 22S) three times.
3.
Combine equal parts of GMPCPP and Taxol-stabilized microtubules at high
concentration (5 mg/ml).
4.
Incubate the microtubules together for 2-24 h at room temperature to enable end-
to-end annealing.
13.1.2.2
Experimental chamber
We perform all biophysical assays in microscope flow chambers with microtubules
immobilized on the surface of a coverslip. This description is similar to that found in
Dixit and Ross (2010)
.
13.1.2.2.1
Buffers, reagents, and equipment
13.1.2.2.1.1
Buffers
PEM-100
13.1.2.2.1.2
Reagents
Monoclonal antibeta tubulin clone TUB 2.1 (Sigma, T5201-200UL,
St. Louis, MO)
5% Pluronic F127 (Sigma, P2443-250G, St. Louis, MO)
Silanized coverslip (22
30
1.5 mm, Thermo Fisher Scientific, Catalog
number 12-544-A, Agawam, MA)
Cover glass (25
1 mm, Thermo Fisher Scientific, Catalog number
12-544-4, Agawam, MA)
Double-stick tape (Scotch 3M, St. Paul, MN)
Kimwipes or filter paper
75
13.1.2.2.2
Detailed procedures
1.
Flow chambers are built by attaching parallel double-sided tape strips to a
glass slide. A silanized glass coverslip is attached to the glass slide with
double-sided tape. The procedure to create silanized coverslips is detailed in
Dixit and Ross (2010)
. In this way, a channel 0.1 mm deep, 3 mm wide, and
30 mm long is created with a volume of approximately 13
m
l(
Fig. 13.3
). To
