Biology Reference
In-Depth Information
depolymerization rate. Calf-brain tubulin self-polymerizes at a
C
r
of 3.3
Mwheninthe
described GAB buffer, so-called favorable conditions (
Buey et al., 2005
). The
C
r
will
change in different conditions and is decreased upon addition of anMSA. The decrease
canbeused tomeasure the assembly inductionpower of anMSA. StrongMSAs promote
MT assembly in conditions that are “hostile” to tubulin assembly, such as in PEDTA
buffer that lacks glycerol. In this buffer, tubulin is unable to assemble unless a strong
MSA is present, or the tubulin is at high concentrations (
m
M) (
D´az,
Menendez, & Andreu, 1993
). Determination of the potency of an MSA involves using
a fixed concentration of tubulin and altering the concentration of theMSA. As theMSA
concentration increases, the supernatant tubulin concentration decreases as MTs form.
200
m
>
19.2.1.1
Microtubule assembly in glycerol buffer
In glycerol buffer, strong and weak MSAs can decrease the
C
r
of tubulin for self-
assembly.
1.
Prepare 20 mg of tubulin in GAB buffer as in
Section 1.1
,add6mMMgCl
2
,
and then increase the concentration of GTP to 1 mM to create assembly conditions.
2.
Incubate samples of 15 and 25
m
m
M ligand to ensure
site saturation (or equivalent volume of DMSO) at 37
C for 30-40 min. For a
blank, use buffer with 15
M tubulin with 20 and 30
M MSA. Use a known MSA as a positive control
(decrease Cr) and a destabilizing agent as a negative control (increase
C
r
).
3.
Separate the MTs by ultracentrifugation (20 min, 37
C). Collect the supernatant
by pipet and dilute 50
m
m
L in 450
m
L of 10 mM NaPi, 1% SDS buffer, pH 7.0.
4.
To the pellet, add 200
m
L of 10 mMNaPi, 1% SDS buffer, pH 7.0 and then dilute
L GAB. (This
ensures that all samples are in the same sample buffer for the standard curve).
5.
Prepare a tubulin standard curve in GAB buffer with tubulin concentrations
ranging from 0 to 20
50
m
L in 400
m
L buffer (10 mM NaPi, 1% SDS pH 7.0) and 50
m
m
M. Dilute 50
m
L of each sample in 450
m
L of 10 mMNaPi,
1% SDS buffer, pH 7.0.
6.
Determine the tubulin concentration of the samples fluorometrically
(
323 nm) using a spectrofluorometer.
7.
Subtract the blank and determine the concentration of tubulin in the supernatants
and pellets from the standard curve. The supernatant and pellet concentrations
should add up to approximately give the total tubulin concentration in the sample.
The
C
r
of tubulin required for assembly changes between different batches of
protein. In order to be able to compare data obtained from different tubulin
batches, the
C
r
data obtained need to be normalized. To do so, determine the
C
r
data in the presence of DMSO, divide all data by this value, and multiply by
3.3
l
exc
¼
280 nm and
l
em
¼
10
6
. This makes the
C
r
value in the presence of the vehicle equal to the
reference value of 3.3
M. The
C
r
of the test ligand is then equal to the tubulin
concentration in the supernatant of that sample.
Using this method, tubulin in the presence of ZMP was found to have a
C
r
of
0.81
m
M, similar to that found in the presence of docetaxel; whereas in the presence
of dactylolide tubulin had a
C
r
of 2.10
m
m
M(
Field et al., 2012
;
Fig. 19.1
A).
