Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 22)

PROTEAN i12 IEF System
The PROTEAN i12 IEF system allows multistep runs
at durations set by either time or Vh. Recommended
starting electrical conditions and voltage ramping
options are provided in Part II of this guide; however,
sometimes the number of Vh required to complete a
run must be determined empirically in a time course
experiment. The optimum Vh depends on the
sample and the composition of the sample solution
as well as the pH gradient of the IPG strip. A more
complex sample or different sample buffer might
change the Vh required. The time needed to achieve
the programmed Vh depends on the pH range
of the IPG strip as well as sample and buffer
characteristics.
Other IEF cells only support running identical pH
gradients and similar samples in batches because
the programmed current and voltage are spread
across all lanes. If different pH ranges or samples
with varying conductivity are run at the same time,
the electrical conditions experienced by individual
IPG strips are different. This may expose some
strips to more or less current than desired, since
the total current limit is averaged over the tray.
The individual lane control provided by the
PROTEAN i12 IEF cell, however, ensures that
the current limit is not exceeded in any IPG strip,
even in situations where conductivity differs
significantly among samples run at the same time.
The PROTEAN i12 IEF cell also allows each lane
to be programmed individually, making it possible
to run different protocols in different lanes.
The flexibility of this system allows running different
experiments at once or varying conditions within
an experiment to allow optimization in fewer runs.
The system also results in better reproducibility
because focusing conditions are not influenced
by other samples in the run.
PROTEAN i12 IEF Cell and Accessories
40 41
2-D Electrophoresis Guide Theory and Product Selection Chapter 3: The First Dimension: Isoelectric Focusing (IEF)
Power Conditions for IEF
The pH gradient and the electric field strength both
influence the time required to reach steady state and
the resolution of the separation. The electric field
strength is determined by the length of the IPG strip
and the applied electrical field. In general, narrow
pH ranges yield higher resolution and require higher
voltages and more time to reach a steady state.
Longer IPG strips can withstand higher programmed
voltages and require an increased number of volt-
hours (Vh) for proper focusing. Vh are the product of
voltage and time. Because the actual voltage reached
is current dependent and the maximum programmed
voltage may not be reached, programming the IEF
cell with Vh can better ensure that samples receive
a consistent number of volts.
The electrical conductivity of the system changes
during an IEF run. At the beginning, the current is
relatively high because of the large number of charge
carriers present. As the proteins and ampholytes
move toward their pIs, and as ionic contaminants
move to the ends of the IPG strip, the current
gradually decreases. When the proteins reach their
final positions in the pH gradient, there is little ionic
movement in the system; the voltage reaches a
maximum, and the current reaches a minimum.
Focusing should occur with a gradual increase in
voltage followed by a prolonged focusing phase at the
maximum voltage advisable for the IPG strip length
used and until a set number of Vh have accumulated.
The optimum duration depends on the length of the
IPG strip and the pH gradient. Current is generally
limited to 50 µA per IPG strip, and a streamlined one-
step protocol is adequate in most circumstances,
as the voltage will rise gradually without the need for
a phased focusing protocol with programmed voltage
ramping. A more gradual focusing protocol may be
advisable in circumstances of heavy protein load,
for some narrow- and micro-range IPG strips,
or when there are high levels of charged contaminants
present. Since the duration of the prolonged focusing
phase is specified in Vh, the actual duration may vary
depending on the average voltage during focusing.
Focusing may conclude at different times for IPG
strips run at the same time with the same protocol.
It is important, therefore, to include a hold step during
which the IPG strip is held at a relatively low voltage to
maintain focusing until the IPG strip can be removed
from the instrument.
Electrical current generates heat, which limits the
magnitude of the electric field that can be applied and
the ionic strength of the solutions that can be used.
Thin gels dissipate heat better than thick gels and thus
better withstand the high voltage that offers higher
resolution. Also, as mentioned above, the current
drops to a constant low value as focusing reaches
a steady state.