Troubleshooting - GE HEALTHCARE Ettan DALTsix User Manual

9

Troubleshooting

Electrical and mechanical
symptom
No current at start of run
Buffer not circulating
Gel casting
symptom
Gel caster leaks
Incomplete gel polymerization
Gel is too soft, too brittle, or white
Gel exhibits swirls
Dye front curves up (smiles)
Vertical protein streaks
Gels cast simultaneously are different sizes
Gradient gels-uneven layering
Spots skewed or distorted
Heavy background after silver staining
Unusually slow or fast run
Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC
possible causes
Insufficient volume of buffer in upper reservoir.
Pump is not primed.
Pump is off.
Pump is broken.
possible solutions
Apply a light film of GelSeal compound to the foam gasket on the front plate before clamping and casting.
Check the foam gasket for cracks or nicks and replace if necessary.
If the stack is too thick, the front plate may not seat firmly against the
gasket. Remove one or more of the filler sheets until the gasket seals.
Use only recent stocks of the highest quality reagents.
If the dry ammonium persulfate does not crackle when water is added to it, replace with fresh reagent.
Use fresh ammonium persulfate.
Solutions of extreme pH may not polymerize.
Degas the monomer solution. Oxygen inhibits polymerization.
Increase both ammonium persulfate and TEMED by 30–50%.
Adjust the gel solution temperature to a minimum of 20 ºC.
Check and adjust crosslinker concentration. Standard SDS gels should have a crosslinker concentration of
2.6% (%C = (g bis × 100)/(g monomer + g bis)).
Make up fresh acrylamide stock solution.
If gel polymerized too fast (<10 min), reduce the concentration of catalyst (APS and TEMED) by 25%.
If gel polymerized too slowly (>50 min), increase the concentration of catalyst (APS and TEMED) by 50%.
Make up fresh acrylamide stock solution.
Check circulation of the buffer.
Pre-chill the buffer.
Decrease power, voltage, or current.
IPG strip not properly placed on gel surface. Make sure IPG strip contacts the gel surface uniformly along its
entire length. Avoid gouging the surface of the separating gel.
Include iodoacetamide equilibration step for IPG strip.
Allow the solution to settle, or reach equilibrium, before applying the overlay.
Apply equal amounts of overlay solution to each gel.
Apply overlay as quickly as possible.
Add sucrose [15% (w/v)] or glycerol (25% (v/v)) to the high-percent monomer solution.
Add a very small amount of bromophenol blue to the high-percent monomer solution to track gradient
formation. Excessive bromophenol blue will inhibit polymerization.
Gels run too fast – uneven migration. Run at a lower power setting. Use a two-step program: start at a low
power setting until the proteins enter the gel, then increase the power for the remainder of the run.
Uneven gel surface. Overlay the running gel with water-saturated butanol before polymerization begins to
avoid forming an uneven gel surface.
Uneven gel polymerization or gradient formation.
Use reagents of the highest purity, preferably electrophoresis grade.
Use deionized, double distilled water.
Check for leaks. All plates, spacers, and gaskets must be clean, dry, and free of grease.
Check the UBC. It should be free of nicks or tears.
Check the pH of the buffer. If the pH is wrong, make fresh buffer, do not back-titrate.
Check recipes, gel concentrations, and buffer dilutions. (For example, do not use Tris·HCl in place of Tris base
for the electrophoresis buffer.)
Discard older acrylamide solutions and use only reagents of highest quality.
Only use freshly deionized urea of highest quality.
Adjust power, current, or voltage.
Troubleshooting 9
possible solutions
Ensure that the unit contains enough buffer to
contact the upper electrode.
Turn pump off and on to purge air bubbles.
Turn on pump.
Service call.
29