Hoefer SE640 - Manual (Page 28)

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Separating the sample
Electrophoresis parameters for discontinuous
polyacrylamide gels
Gels may be run at either constant current or
constant voltage settings. A constant current set-
ting is traditionally used with a discontinuous
buffer system so that the rate of electrophoretic
migration remains unchanged throughout the
run. Under these conditions, voltage increases
as the run proceeds. A lower current setting is
recommended for higher resolution. The optimal
current level must be determined empirically;
the main factors that must be balanced include
the gel concentration and migration speed, and
the resulting Joule heating and band distortion.
Table 2 lists starting point guidelines and adjust-
ments for gel thickness, number of gels, and
migration rate.
Current
Current acts on the total cross-section area of
all the gels because the gels are connected in
parallel in the electrical circuit. Thus the current
setting for one gel must be multiplied by the
number of gels of the same gel thickness that
are run simultaneously. For a gel 1.5 mm thick,
we suggest a starting current setting of 25 mA.
(Two 1.5 mm gels = 50 mA.)
Voltage
The starting voltage for a 1.5 mm slab gel con-
nected to a power supply set to 25 mA is usually
80 to 90 V (using the SE600 with a Laemmli
discontinuous buffer system for SDS gels). The
final voltage can typically range from 220 to
400 V, depending on the length of the gel. (See
Table 2.)
Note: All SE600 series models
use 18-cm wide plates. The
gel thickness determines the
cross section (and current
requirement). The length
of the plate determines the
running time.
Table 2: Laemmli buffer
system starting point
guidelines
Gel thickness* 1.5 mm
Current per gel † 25 mA
constant
current
Starting voltage ‡ 80 – 90 V
Final voltage 220 – 400 V
* Thicker or thinner gels require propor-
tionally more or less current. For
example, a 0.75 mm gel, which is half
as thick as a 1.5 mm gel, requires half
as much current, or 12.5 mA.
† The current must be multiplied by
the number of gels. For instance, if
two club sandwiches are installed, the
four gels require four times as much
current. The current can be increased
for faster runs though overheating will
eventually become a problem, and it
can be decreased for slower overnight
runs.
‡ At 25 mA per gel.