Chemistry Reference
In-Depth Information
Figure 4.3 (A) Schematic diagram showing the
incorporation of channel proteins into a
membrane. The anti-BK channel antibodies were
labeled with Cy5 dye molecules. The vesicles
were incubated with labeled antibodies. After
removal of unreacted antibodies by
ultracentrifugation, the vesicles were added
directly to the bilayer through a fine glass
capillary. The channel protein was transferred
into the planar bilayer by fusion between the
vesicular membrane and the planar bilayer.
(B) The specificity of anti-BK antibody
investigated using SDS-PAGE and Western
blotting. Column (a) shows CBB staining of SDS-
PAGE (420%). Columns (b) and (c) represent
Western blots produced with anti-BK antibody
and secondary antibody respectively as the
negative control. For the positive control, the
fusion protein from Schistosoma japonicum
glutathione-S-transferase (GST) and the
C-terminal part of the mouse
subunit from
Alomone labs (Jerusalem, Israel) were used in
lane 3 in each column. The 37-kDa control fusion
protein only stained intensely when treated with
anti-BK antibody. The 70-kDa protein which is
stained in both in b and c indicates that this
molecule is a non-specific protein. In the
sarcolemmmal vesicle fraction, the 120-kDa
protein was stained intensely when exposed to
the anti-BK antibody (column b, lane 2). A prote
in of this size corresponds with the expectedMW
(125 kDa) of BK [10]. As shown in column a,
the sarcolemmal vesicle fraction contained
very little 125-kDa BK protein. These results
confirm the specificity of this anti-BK antibody
against BK in the vesicle fraction.
a
and the most suitable ionic concentrations had to be determined for every vesicle
preparation.
A bright spot appeared just before the current across the membrane began to
fluctuate as shown in Figure 4.4A. After incorporation, the spot moved rapidly within
the bilayer as shown in Figure 4.4B. The diffusion constant, D, was calculated to be
3.0
10
8
cm
2
/s (n
5) which agrees well with the value for a channel moving
freely in a membrane. This result shows that the
fluorescently labeled channel
proteins in the vesicular membrane moved thermally in the solution and when in
close proximity to the horizontal planar bilayer became incorporated into the bilayer
through vesicle fusion. In other words, the bright spot shown in Figure 4.4
1.5
ΒΌ
