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Note: With Coomassie Blue
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.
Note: Once the samples are in the
wells, take care to not jar the sand-
wiches so that the samples are not
spilled or mixed.
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p16
Sample preparation and loading
The sample can be loaded either while the sandwich is in the
caster or after the upper buffer chamber is attached. When loading
™
it is
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 detection 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 stack-
ing gel is used. In a discontinuous buffer system, the zone into
which each molecular species migrates is sharpened by the stack-
ing gel, so the sample need not be as concentrated.
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.
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 buf-
fer and high-purity water to achieve the desired concentration.
Heat the tube in boiling water for 90 s, 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 min. Store unused sample
at 4 °C.
Underlay the sample into the wells using a fine-tipped microsy-
ringe or gel-loading pipette tip.
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