Hoefer SE640 User Manual
Page 36
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p28
Poor band
resolution
Running
conditions
Begin electrophoresis as soon as the sample is loaded to pre-
vent 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.
Sample
preparation
Store sample on ice before it is denatured.
Dialyze or desalt the sample.
Heat samples in SDS sample buffer for no more than
1–2 min at 100 °C to improve dissociation of subunits.
Store on ice after heating.
Adjust the sample volume or concentration.
Add more mercaptoethanol or dithiothreitol; check sample
treatment.
Add protease inhibitors such as PMSF if necessary to pre-
vent proteolytic degradation of sample.
Increase glycerol or sucrose to increase sample density.
Store samples to be frozen in aliquots to avoid repeated
freeze-thawing. Store at -40 to -80 °C.
problem
possible cause
remedy