Hoefer SE640 - Manual (Page 35)

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Bands are
skewed or
distorted Incomplete gel
preparation and
polymerization Degas the stacking-gel solution and avoid trapping air bub-
bles under the comb teeth.
Irregular interface
between stacking
and running gels Overlay the running gel with water-saturated butanol before
polymerization begins, to avoid forming an uneven gel sur-
face.
Sample preparation Dialyze or desalt the sample.
Stained sample collects:
Near the buffer
front Gel concentration Molecules are not sufficiently restricted by the resolving gel
pore size: increase the %T.
Degradation Proteins may be degraded by endogenous proteases: use pro-
tease inhibitors during the isolation step.
Near the top of
the gel when
the buffer front
has reached the
bottom Gel concentration The gel pore size is too small: decrease the %T of the resolv-
ing (or stacking) gel.
Precipitation The protein has precipitated. Heat the sample at a lower
temperature (70 °C or less) for 1–2 min.
At both top
and bottom of
the gel Gel concentration The molecular weight range of the sample requires an acryl-
amide concentration gradient to resolve the full range of
protein sizes.
problem possible cause remedy
Tracking dye
doesn’t sharpen
into a concentrated
zone in the
stacking gel Poor stacking Pour a taller stacking gel. (For best results, allow a
stacking-gel height of 2.5 times the height of the sample in
the well.)
Reagent quality Dispose of outdated acrylamide solutions and use only the
highest grade of acrylamide.
Sample preparation When preparing samples, avoid using solutions with high salt
concentrations.