Biology Reference
In-Depth Information
14.2.2 Materials
1. Tubulin (either commercial or lab purified). Tubulin is available commercially
(e.g., Sigma-Aldrich, St. Louis, MO; Cytoskeleton, Boulder, CO), but
purification of MTP or tubulin from bovine or rat brain is not difficult and
protocols are available ( Andreu, 2007; Hamel & Lin, 1981; Sackett, Knipling, &
Wolff, 1991 ), but not discussed here. Tubulin can also be readily purified from
nonneural sources for these assays ( Sackett, Werbovetz, & Morrissette, 2010 ).
Whatever the source, tubulin is usually stored at
80 C in aliquots in order to
minimize the numbers of freeze-thaw cycles. We purify our tubulin in-house,
store it at high concentration (25 mg/ml) in GTP-free PM buffer, and limit
freeze-thaw cycles to two thaws.
2. PM buffer
0.1 M Pipes, 1 mM MgCl 2 , pH 6.9. Pipes free acid is not very
soluble in water, so is titrated into solution, and then to pH 6.9, with NaOH. A PM
buffer with 1.0 M TMAO should be used when assaying depolymerizing agents,
and 0.8 M TMAO should be used for stabilizing agents.
3. GTP stock solution
¼
¼
0.1 M in PM buffer.
4. DMSO.
5. Podophyllotoxin: Stock solution
¼
4.1 mg/ml DMSO gives 10 mM. Working
solution
1 mM in DMSO. This is an inexpensive and effective inhibitor of
polymerization.
¼
14.3 METHODS
14.3.1 Preparation of tubulin
Tubulin may be commercial or lab prepared. Once an aliquot is thawed for an
experiment, a working stock is prepared at 2.5 mg/ml in PM buffer and kept on
ice. A brief high-speed spin (5 min at 20,000
g or top speed in a 4 C microcen-
trifuge or table-top ultracentrifuge) is performed to remove any aggregates, and
the top 90% of the solution is removed to a new tube. With good tubulin preparations,
this results in the loss of very little material and, for many purposes, can be skipped
once this is verified. The tube is kept on ice.
14.3.2 Turbidity assay in cuvettes
The cuvette of choice is a microvolume, self-masking cuvette with a working volume
of 50
m
m
l, though we usually use 60
l, to avoid a meniscus in the light path. The final
m
sample will be 60
l, 1.25 mg/ml tubulin, 0.5 mM GTP, 5% (v/v) DMSO, 1.0 M
TMAO, in PM buffer. Tubulin and GTP concentrations may be altered as desired
for particular experiments. Lowering the TMAO concentration (e.g., to 0.8 M) will
raise the C C and make the reaction more suitable for assay of polymerization-
promoting compounds (such as paclitaxel). The sample is prepared in two halves:
tubulin plus any test drug in one half (both at twice the desired final concentration)
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