Biology Reference
In-Depth Information
19.1.3
Software for data analysis
Equigra v5.0 (F. D
´
az, unpublished) for Flutax competition assay analysis (available
from the author). This program fits the experimental data by least-squares to the equi-
librium binding constant of the ligand under investigation, using the known values for
a reference ligand. Instructions for program use can be found in
D´az and Buey (2007)
.
The binding constant of the test ligand,
K
(1), can be determined from the known
values of the binding constant of Flutax,
K
(
r
), and the concentrations of binding sites,
Flutax and test ligand by solving the simultaneous mass action equations (
D´az &
Buey, 2007
):
K
(1)
¼
[Ligand]
bound
/[Sites]
free
*[Ligand]
free
K
(
r
)
[Flutax]
bound
/[Sites]
free
*[Flutax]
free
[Flutax]
free
¼
¼
[Flutax]
total
[Flutax]
bound
[Ligand]
free
¼
[Ligand]
total
[Ligand]
bound
[Sites]
free
¼
[Sites]
total
[Ligand]
bound
[Flutax]
bound
19.1.4
Ultracentrifugation
In all experiments, ultracentrifugation is performed using a Beckman Optima TLX
ultracentrifuge employing a TLA 120.2 (2-mL tubes) or TLA 100 (200-
m
L tubes)
g
(temperature and running time is experiment-dependent). Alter-
natively, other ultracentrifuges and rotors can be used providing the parameters
are kept the same as described in these experiments. For example, we have used a
Beckman Coulter Optima
™
L-100 XP ultracentrifuge, employing a type 70.1 Ti rotor,
using Delrin adaptors for thick-walled 4.0-mL polycarbonate tubes (Beckman
Coulter). If only large rotors and tubes are available, mineral oil can be used to make
up the final volume to the minimum required for centrifugation, and the aqueous
sample can then be removed from under the oil layer after centrifugation.
rotor at 90,000
19.2
METHODS AND RESULTS
The first task to characterize a suspected MSA is to confirm that it is a true MSA and
determine how potent it is
in vitro
. This provides information on the assay conditions
needed for further experiments. Strong MSAs can induce tubulin assembly in con-
ditions that tubulin on its own is not able to assemble or assembles very slowly. A
weaker MSA should at least be able to enhance tubulin assembly in conditions where
tubulin is already able to assemble. Ligand-induced MTs also need to be confirmed
visually by electron microscopy.
19.2.1
Induction of microtubule assembly
Tubulin polymerization can be easily monitored by quantifying polymer amounts in a
pellet following centrifugation. Tubulin assembles into MTs from a critical concentra-
tion (
C
r
), the concentration of tubulin at which polymerization rate overcomes
