Hoefer SE250 - Manual (Page 24)

problem solution
Stained sample collects: Near the buffer front:
• Protein is not sufficiently restricted by the resolving
gel; increase the % T.
Near the top of the gel when the buffer front has
reached the bottom:
• The gel pore size is too small. Decrease the % T of
the resolving gel.
• The protein has precipitated. Heat the sample at a
lower temperature (70 °C or less) for 1–2 minutes.
Poor band resolution • Use only the highest quality reagents.
• Conduct the separation at a lower current or
voltage setting.
• Dialyze or desalt the sample.
• Reduce the sample volume or concentration.
• Only use freshly deionized urea.
• Improve dissociation of subunits by heating sample
in SDS sample buffer 1–2 minutes at 100 °C.
• Add more mercaptoethanol or dithiothreitol; check
sample treatment.
• Only use gels that were recently prepared.
• Check pH values of the separating and stacking gel
solutions. Do not back-titrate buffers.
Sample preparation:
• Heat samples for no more than 1–2 minutes at
100 °C. Store on ice after heating.
• Store sample on ice before it is denatured.
• Add protease inhibitors if necessary to prevent
proteolytic degradation of sample.
• Store samples to be frozen in aliquots to prevent
repeated freezing and thawing. (Store at -40 °C to
-80 °C.)
Bromophenol blue doesn’t sharpen • Pour a taller stacking gel. (For best results, allow a
into a concentrated zone in the • stacking gel height of 2.5 times the height of the
stacking gel • sample in the well.)
• Dispose of outdated acrylamide solutions and use
only the highest grade of acrylamide.
• When preparing samples, avoid using solutions with
a high sodium or potassium concentration.
•
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