Hoefer SE400 - Manual (Page 35)

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p27
problem/possible cause remedy
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 protease 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
resolving (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
acrylamide concentration gradient to resolve the full range
of protein sizes.
Poor band resolution
Running conditions Begin electrophoresis as soon as the sample is loaded to
prevent low molecular weight species from diffusing.
Conduct the separation at a lower current or voltage setting to
reduce Joule heating.
Reagent quality Use only the highest-quality reagents.
Poor stacking Use only gels that were recently prepared.
Add a stacking gel or increase height of the stacking gel.
Prepare the resolving-gel surface by first rinsing it with
stacking-gel monomer before pouring the stacking gel to
ensure continuity between the gels.
Check pH values of the resolving- and stacking-gel solutions.
Do not back-titrate buffers.
Incomplete gel polymerization Allow gel to polymerize fully.