Hoefer SE400 - Manual (Page 34)

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problem/possible cause remedy
Upper buffer chamber leaks
Mis-aligned parts Check that the glass plates, spacers, and clamps are aligned
and fit snugly into the upper chamber gasket.
Check that both gaskets are centered and that the positioning
ridges fit inside the grooves.
Dirty or Check that the gasket is not damaged or pinched. Replace if
damaged components necessary. Check that the upper buffer chamber is not warped
from prior exposure to excessive heat.
Dye front curves up (smiles) at edges
Excessive heat Prechill the buffer.
Decrease the current or voltage setting.
Run the gel in the cold room.
Protein streaks vertically
Particulates in sample Centrifuge or filter sample before loading to remove particulates.
Overloading Load less sample.
Degradation Add protease inhibitor such as PMSF.
Unusually slow (or fast) run
Current leakage around gel Check for leaks; all plates and spacers must be aligned
and free of grease and cracks.
Sample or reagent preparation If the required pH of a solution is overshot, do not
back-titrate. Discard and prepare fresh buffer.
Check recipes, gel concentrations, and buffer dilution.
(For instance, do not use Tris-HCl instead of Tris for Laemmli
tank buffer.)
Decrease the salt concentration of samples.
Reagent quality Dispose of older acrylamide solutions and use only stock of
the highest quality.
Use only freshly deionized urea.
Voltage or current settings To increase or decrease the migration rate, adjust the voltage
or current by 25–50%.
Bands are skewed or distorted
Incomplete gel preparation Degas the stacking-gel solution and avoid trapping air bubbles
and polymerization under the comb teeth.
Irregular interface between Overlay the running gel with water-saturated butanol before
stacking and running gels polymerization begins, to avoid forming an uneven gel surface.
Sample preparation Dialyze or desalt the sample.