Biology Reference
In-Depth Information
FIGURE 1.6
Monomeric quantal brightness determination using pharmacological agents that block
oligomerization and induce monomeric conformation.
1.3.2.3
Determining the monomeric QB by using pharmacological agents
that block oligomerization and induce monomeric conformation
In our studies with RTKs EGFR and TrkB, the inhibitor AG1478 (
Levitzki & Gazit,
1995
) was used to block oligomerization and force a monomeric RTK population. In
our hands, the use of this inhibitor with CHO-EGFR-GFP cells revealed that follow-
ing growth factor starvation, most of EGFR-GFP were in monomeric conformation
(
Fig. 1.6
).
In summary, any sample or region of a sample that contains a vast majority of
monomers that are labeled with the same fluorescent probe, when imaged with
the same microscope using the same experimental settings (e.g., laser intensity,
PMT voltage, pixel dwell times, and pixel size), can be used to obtain an estimate
of the monomeric QB.
1.3.3
Image acquisitions
Optimal CLSM conditions should be adjusted for each experimental design. CLSM
settings should be chosen to achieve a good signal, that is, within the linear dynamic
range of the detector and not saturated. Laser intensity and PMT gain are adjusted to
minimize pixel saturation and photobleaching. Once found, image collection param-
eters (settings of laser power, filters, dichroic mirrors, polarization voltage, scan
speed and pixel dwell time, PMT gain, laser intensity detector, and pinhole) are kept
