3.4 Sample preparation and loading
If wells are already in place, skip to step 2.
If applicable, cast the stacking gel in the unit.
Calculate the stacking gel monomer solution volume:
measure the distance, in cm, from the top of the
resolving gel to the notch in the alumina plate. (This
should be at least 2 cm—more if the sample depth
in the well is unusually high.) Multiply this distance
by the gel width (8.3 cm) and the gel thickness (cm).
This product is the required volume in ml.
Deaerate the stacking gel monomer solution, add
catalyst and initiator and then pour. Use a pipette
to deliver the solution into one corner of the plate,
taking care not to trap any bubbles. Insert a comb (at
a slight angle to prevent trapping air) into the sand-
wich, allowing the comb sides to rest on the spacers.
Prepare the sample. Increase liquid sample density
with 10% glycerol or sucrose. Add a tracking dye such
as phenol red or bromophenol blue.
For SDS protein gels, use 2X treatment buffer to
denature both liquid and dry samples in a test tube.
To liquid protein solutions
, add an equal volume of 2X
buffer.
To dry protein samples
, add equal volumes of
buffer and ddH
2
O to achieve the desired concentra-
tion. Heat the tube in boiling water for 90 seconds,
then chill it in ice until ready to use. Treated samples
can be stored frozen for future runs. (Store at -40 °C
to -80 °C.)
To aid in loading samples, wet the well-locating decal
and apply it to the front of the glass plate so that the
appropriate edge outlines the sample wells.
Note:
The side wells for standards of a preparative
comb correspond to the outer-most wells formed by
the 10-well comb.
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Note:
Stacking gel resolution
is optimal when poured just
before electrophoresis.