Biology Reference
In-Depth Information
4.
Incubate the samples (2 h, 37
C) and vacuum dry. Store the samples (
20
C)
until analysis.
19.2.6.1.2
Stabilized cross-linked microtubules
If the MSA is a weak inducer of MT assembly, use cross-linked stabilized MTs. This
allows the ligand to bind straight to preformed MTs.
1.
Incubate 20
M ligand or an equivalent
volume of DMSO in GAB-0.1 mM GTP buffer for 4 h at 25
C (4 h should be
sufficient time for weaker compounds to react).
2.
Pellet the MTs by centrifugation (20 min, 25
C) and discard the supernatant.
Resuspend the pellet in 200
m
M stabilized taxoid sites with 25
m
m
Lof50mMNH
4
HCO
3
and process the samples as
per steps 3-4 above.
19.2.6.1.3
Unassembled tubulin
It is important to check if the ligand interacts with a different residue in oligomeric or
unassembled tubulin compared to MTs.
1.
Prepare 20 mg tubulin in PEDTA buffer as described in
Section 1.1
.
2.
Add 1.5 mM MgCl
2
for oligomer formation. For dimeric tubulin, omit
this step.
3.
Incubate 20
M ligand in this buffer at 25
C for 4 h or
overnight, depending on how strong a binding agent the test MSA is.
4.
Collect the tubulin supernatant by centrifugation (20 min, 25
C), add 20
m
M tubulin with 25
m
m
Lof
LofNH
4
HCO
3
.
5.
Incubate the samples at 37
C for 2 h and vacuum dry overnight. Store the
samples at
the supernatant to 1
m
g/
m
L trypsin, and then add 20
m
20
C until analysis.
19.2.6.1.4
Compounds
To determine the best ion tracer fragments, prepare compounds without protein
(1-20 mM). Keep at
80
C for 1 h before lyophilizing overnight (
Fig. 19.5
).
19.2.7
Visualization of covalent binding in cells
Covalent binding
in vitro
needs to be confirmed in a cellular setting to ensure that the
same mechanism of binding is occurring. For taxoid binding site ligands, this can be
carried out using Flutax and conventional fluorescence or confocal microscopy. Al-
ternatively, MS of cellular tubulin can be used to confirm the covalent binding in
cells, as described by
Buey et al. (2007)
.
Using Flutax, covalent binding can be visualized in cells using simple competi-
tion methods. Known taxoid site MSAs can be used as positive controls and those
that bind the laulimalide/peloruside site as negative controls. In the case of a revers-
ible taxoid site ligand, when added in excess over Flutax, there will be no Flutax
staining of the MTs. When the Flutax is given in excess, however, the MTs will
be stained as only one of the two ligands can bind the site at any one time, and Flutax
in excess outcompetes the test MSA. Using an MSA that binds the laulimalide/
