Hoefer SE250 - Manual (Page 17)

3.4 Sample preparation and loading
1
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 sandwich,
allowing the comb sides to rest on the spacers.
Overlay each gel with a thin layer of water-saturated
n-butanol, water, or diluted gel buffer to prevent gel
exposure to oxygen. Slowly deliver the overlay solution
from a glass syringe fitted with a 22-gauge needle.
Apply the solution near the spacer at the side of
the sandwich and allow it to flow across the surface
unaided. Allow a minimum of one hour for the gel
to polymerize.
2
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
concentration. 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.)
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p9
Note: Stacking gel resolution is
optimal when poured just before
electrophoresis.