Biology Reference
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19.2.1.2 Microtubule assembly in PEDTA buffer
To determine if a compound is a strong MSA, its ability to induce tubulin assembly in
conditions that are unfavorable for assembly is determined. In these conditions, weak
MSAs such as cyclostreptin cannot induce MT assembly.
1. Prepare 20 mg tubulin in PEDTA buffer as described in Section 1.1 . Add 6 mM
MgCl 2 to the buffer and increase the concentration of GTP to 1 mM.
2. Prepare samples of 20
M tubulin with increasing concentrations of MSA and
incubate at 37 C for 30-40 min. Use a sample with 0
m
m
m
M tubulin and 15
M
MSA as a blank.
3. Separate the supernatant from the pellet by ultracentrifugation (20 min, 37 C).
4. Collect the supernatant and resuspend the pellet in 10 mM NaPi, 1% SDS buffer,
pH 7.0.
5. Dilute the samples, prepare a standard curve, and measure tubulin polymerization
fluorometrically as in step 6 above.
6. Analyze the data as in step 7 above but do not normalize. A decreasing amount of
tubulin in the supernatant will be seen as MSA concentration increases. When
this value stabilizes, it represents the C r .
The C r for tubulin in the presence of ZMP in these conditions is 4.1
m
M( Fig. 19.1 B).
FIGURE 19.1
Induction of microtubule assembly by ZMP. (A) Induction of MT assembly by ZMP in
conditions favorable for assembly. In these conditions, the C r for self-induced assembly is
3.3
m
M (DMSO bar and dashed line). Both docetaxel and ZMP significantly decrease the C r to
0.65
M, respectively. Dactylolide, less potent than ZMP, is able to decrease
the C r to 2.10. (B) Induction of assembly by ZMP in conditions unfavorable to tubulin
assembly. In these conditions, ZMP has a C r of 4.1
m
M and 0.81
m
m
M. Normally in these conditions, the
tubulin concentration required for assembly is
M.
This figure was modified from Field et al. (2012) with permission from the publisher.
200
m
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