Hoefer SE400 - Manual (Page 23)

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2.3 Sample preparation
The amount of sample loaded depends on the
thickness of the gel, the sensitivity of the detec-
tion method used, and the amount of sample
expected in each band. In a continuous buffer
system, the protein sample should be relatively
concentrated because no stacking gel is used.
In a discontinuous buffer system, the zone into
which each molecular species migrates is sharp-
ened by the stacking gel, so the sample need not
be as concentrated.
1
Prepare the wells. Remove the comb by gently
rocking it side to side and then lifting it straight
up to avoid damaging the well walls. Carefully rinse
each well with electrophoresis buffer to remove
unpolymerized acrylamide and then drain by inverting
the gel sandwich (or caster). Fill each well with
electrophoresis buffer.
2
Prepare the sample. Increase liquid sample density
with 10% glycerol or sucrose. Add a tracking dye such
as phenol red, bromophenol blue, or pyronin Y.
For SDS protein gels, use 2X treatment buffer to
denature both liquid and dry samples in a test tube:
To liquid protein samples, add an equal volume of
2X treatment buffer.
To dry protein samples, add equal volumes of 2X
treatment buffer and deionized water to achieve the
desired concentration.
Heat the tube in boiling water for 90 seconds, then
allow to cool to room temperature. Treated samples
can be stored at -40 to -80 °C for future runs.
Heat membrane proteins to 60 °C for 20 minutes.
Store unused sample at 4 °C.
Table 2. Well volume (µl) per
1 mm depth for each comb size
Comb thickness
(mm)
No. of wells 0.75 1.0 1.5
10 6.2 8.3 12.4
12 5.8 7.7 11.5
15 4.3 5.7 8.6
20 3.1 4.1 6.2
28 2.1 2.7 4.1