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Operation 5
The parameter that should be chosen as the constant and thus control the
electrophoresis depends on the type of electrophoresis. In the case of homogeneous
buffers throughout the system (same electrode and gel buffer), the conductivity is
constant during the electrophoresis. If the conductivity is constant, the voltage will be
proportional to the current and the power to the square of the current, according to (1)
and (2). This means that the result of the electrophoresis will be the same, regardless
which parameter is chosen for constant. For historical and practical reasons, voltage
is most commonly used for regulation. Submarine gel electrophoresis of DNA/RNA
and pulsed field electrophoresis are usually run at constant voltage. SDS-PAGE using
continuous buffer systems is run at constant voltage or current.
For discontinuous buffer systems, the resistance will increase as the electrophoresis
proceeds due to a decrease in conductivity. Running at constant voltage will result in
decreasing current and power. Constant voltage will thus be "safe" in the respect that
the power will not increase and produce more and more heat. On the other hand, the
separation will slow down and impair resolution due to an increased time available for
diffusion. Running at constant power would give a faster electrophoresis and controlled
power, while running at constant current would, at the first sight, seem to be problematic
because of increasing voltage and power.
During discontinuous electrophoresis, however, the voltage is not distributed evenly
across the gel. These gels have a region with low ionic strength that causes a high
electrical field strength. This region increases as the electrophoresis proceeds. This
means that the main part of the voltage is spread over a longer and longer distance
and a higher and higher power is tolerated. This is the reason why constant current is
chosen for such applications. It is, however, recommended to also limit the power as a
precaution against overheating the gel. The power supply will probably switch over to
limiting power at the end of the run due to increased voltage.
The crossing-over between different parameters controlling the electrophoresis
can be illustrated by IEF (isoelectric focusing) using carrier ampholytes. A graphical
representation of the changes in power, voltage and current that may occur during a
typical IEF run is given in Fig 6. Since the pK values of the carrier ampholytes and the
proteins are temperature dependent, IEF must be carried out at a constant temperature.
Therefore cooling of electrophoresis equipment and controlling by power is recom-
mended. The main part of the IEF is thus controlled by power (phase II). The conductivity
is gradually decreasing because the carrier ampholytes and sample will loose their net
charge during the build up of the pH gradient. Thus the current will decrease and the
voltage increase at constant power. During the early stage of the formation of the pH
gradient it is important to limit the current. Otherwise the gradient will be irregularly
shaped (phase I). The conductivity is not constant along the gel so it is important to also
limit the voltage. This means running at constant voltage for the last phase (phase III) to
prevent local overheating.
For detailed information about parameter values, always follow the gel supplier´s
recommendations.
Electrophoresis Power Supply EPS 3501 User Manual 18-1130-21 Edition AD
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