Biology Reference
In-Depth Information
Buffer formulations used for the assembly reaction significantly influence the poly-
merization, both by altering the critical concentration for polymerization and by al-
tering the exact polymer produced—for example, by increasing the production of
sheet polymers in addition to MT. Both the turbidity and the fluorescence methods
are useful for demonstrating the effect of MT-stabilizing or -destabilizing additives.
14.1 BACKGROUND AND THEORY
14.1.1 Background
This chapter presents protocols for the assay of in vitro microtubule (MT) polymer-
ization from purified proteins in defined buffer solutions. Purified tubulin will poly-
merize into MT and other polymers under correct conditions. This was discovered
years ago, partially as a result of using a buffer solution that chelated calcium
( Weisenberg, 1972 ). This reaction has been widely used, not only to understand
MT polymerization per se but also as a means to check the action of other proteins
that alter MT properties or polymerization. Perhaps, its widest application has been
in evaluating the action of MT-active drugs ( Hamel, 2003 ).
Measuring MT polymerization depends on measuring the change in some phys-
ical property that differs between a solution of dimeric tubulin and a solution of poly-
mers like MT. The most obvious of these properties is size. As dimers assemble into
polymers, the solution scatters more light, which can be detected by the increased
optical density measurable by turbidity assay in a spectrophotometer ( Gaskin,
Cantor, & Shelanski, 1974 ). The difference between dimer and polymer size also
allows for the use of sedimentation and filtration assays ( Bollag et al., 1995 ). In ad-
dition to size differences, polymerization also changes binding properties of the pro-
tein so that fluorescent probes such as diamidino-phenylindole (DAPI) that bind to
tubulin increase their emission intensity upon polymerization of tubulin ( Bonne,
Heus´le, Simon, & Pantaloni, 1985; Heusele, Bonne, & Carlier, 1987 ).
Turbidity is the most widely used method for following tubulin polymerization
because it is simple, requires no unusual equipment, and is quantifiable. A number of
papers have described the theory behind these measurements and how to maximize
the information they may yield ( Berne, 1974; Detrich, Jordan, Wilson, & Williams,
1985; Gaskin, 2011; Hall & Minton, 2005 ). It is readily adapted to multiwell plate
assays and indeed is commercially available in this form ( Davis, Martinez, Nelson, &
Middleton, 2010 ). All that is required is a spectrophotometer and an optically clear
vessel (cuvette or multiwell plate) to hold the tubulin sample in the light beam. With
moderate care, the resulting turbidity readings can yield quantitative measurements
of assembly kinetics and steady-state polymer concentrations. Slightly more compli-
cated protocols and analyses will also yield information on polymer form (are these
tubulin polymers really MT?) and reversibility. Some of these are described below.
The drawbacks of a turbidity assay are not always appreciated but may lead to
considerable misinterpretation. MT formation is not the only thing that can cause
an increase in turbidity in a tubulin solution. Addition of HCl can do the same thing,
by precipitating the tubulin in aggregates. It is important to check that the observed
turbidity is due to formation of MT. Other non-MT polymers of tubulin can also
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