Biology Reference
In-Depth Information
FIGURE 17.4
Polymerization of BtubA/B in 20 mM Tris, 300 mM potassium glutamate, 1 mM EGTA, 5 mM
MgCl
2
, 1 mMGTP, pH 7.5, at 25
C, measured by sedimentation. (A) SDS-PAGE of the pellets
and supernatants, sequentially loaded with an electrophoretic shift in the same lanes. (B)
Protein sedimentation measurements (left axis, filled circles, pellet protein concentrations;
the triangles are controls without nucleotide and with GDP), compared with light scattering
values (right axis, empty squares, plateau scattering). The solid line is the least squares
linear fit to the centrifugation data. This BtubA/B was prepared with the polymerization-
depolymerization cycle right after the high-speed centrifugation of the cell lysate (purification
step 1).
polymers and rule out the possible formation of aggregates. As an example,
Fig. 17.5
shows the assembly a BtubA/B chimera in which the M loop of
-tubulin was ex-
changed into BtubB, in comparison with wild-type BtubA/B and microtubules, in
Pipes microtubule assembly buffer with the slowly hydrolysable nucleotide
GMPCPP, examined with light scattering, sedimentation, and electron microscopy.
The repeat distances between monomers in the BtubA/B polymer lattices can be an-
alyzed with diffraction patterns computed from their micrographs (
Martin-Galiano
et al., 2011; Pilhofer et al., 2011; Schlieper et al., 2005
).
b
