Hoefer SE640 - Manual (Page 23)

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p15
Sample preparation and loading
The sample can be loaded either while the sand-
wich is in the caster or after the upper buffer
chamber is attached. When loading samples while
using divider plates, the samples must be loaded
without the upper buffer chamber in place.
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 distilled water 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 solutions, add an equal volume of
2X buffer.
To dry protein samples, add equal volumes of 2X
sample buffer and high purity water to achieve the
desired concentration.
Note: With Coomassie ™ Blue it
is possible to detect 1 µg of
protein in a single band. With
the more sensitive silver stains,
it is possible to detect as little
as 10 ng of protein.