Separating The Sample - Hoefer SE 600 Chroma User Manual

Note: SE 600 Chroma unit uses
18-cm-wide plates. The gel thickness
determines the cross section (and
current requirement) for constant
current runs. The length of the plate
determines the running time.
Table 2:
Laemmli buffer system
starting point guidelines
Gel thickness*
Current per gel †
Starting voltage ‡
Final voltage
*Thicker or thinner gels require
proportionally 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 if active cooling is used and it
can be decreased for slower over-
night runs.
‡ At 25 mA per gel.
•
p22
1.5 mm
25 mA
con-
stant current
80–90 V
220–250 V

Separating the sample

Electrophoresis parameters for
discontinuous polyacrylamide gels
Gels may be run at either constant current or
constant voltage settings. A constant current
mode 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 rec-
ommended 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 distor-
tion. Table 2 lists starting-point guidelines and
adjustments 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 cur-
rent setting for one gel must be multiplied by
the number of gels of the same cross-section run
simultaneously. For a gel 1.5 mm thick, we sug-
gest a starting current setting of 25 mA. (Two
1.5 mm gels = 50 mA.) Note: Cooling may be
required to control Joule heating.
Voltage
The starting voltage for a 1.5 mm slab gel con-
nected to a power supply set to 25 mA is usu-
ally 80–90 V (using the SE 600 Chroma unit