A tubulin polymerization
kit (Cytoskeleton, Denver, CO) was used to evaluate effect of the
pyridine-linked CA-4 analogues on tubulin assembly in vitro.24 (link),25 (link) It is based on the principal that light is scattered by microtubules
to an extent that is proportional to the concentration of the microtubule
polymer. Compounds that interact with tubulin will alter the polymerization
of tubulin, and this can be detected using a spectrophotometer. The
absorbance at 340 nm at 37 °C is monitored. The experimental
procedure of the assay was performed as described in version 8.2 of
the tubulin polymerization assay kit manual. Varying concentrations
of compounds were preincubated with 10 μM bovine brain tubulin
in glutamate buffer at 30 °C and then cooled to 0 °C. After
the addition of 0.4 mM GTP, the mixtures were transferred to 0 °C
cuvettes in a recording spectrophotometer and warmed to 30 °C.
Tubulin assembly was monitored by measuring the optical density at
340 nm using a BioTek Synergy 4 multifunction microplate spectrophotometer.
kit (Cytoskeleton, Denver, CO) was used to evaluate effect of the
pyridine-linked CA-4 analogues on tubulin assembly in vitro.24 (link),25 (link) It is based on the principal that light is scattered by microtubules
to an extent that is proportional to the concentration of the microtubule
polymer. Compounds that interact with tubulin will alter the polymerization
of tubulin, and this can be detected using a spectrophotometer. The
absorbance at 340 nm at 37 °C is monitored. The experimental
procedure of the assay was performed as described in version 8.2 of
the tubulin polymerization assay kit manual. Varying concentrations
of compounds were preincubated with 10 μM bovine brain tubulin
in glutamate buffer at 30 °C and then cooled to 0 °C. After
the addition of 0.4 mM GTP, the mixtures were transferred to 0 °C
cuvettes in a recording spectrophotometer and warmed to 30 °C.
Tubulin assembly was monitored by measuring the optical density at
340 nm using a BioTek Synergy 4 multifunction microplate spectrophotometer.
