Sample Preparation & Gel Electrophoresis Troubleshooting

Electric currents, wires, leads, combs, leaks… so many opportunities for trouble. But we still use gels, because electrophoresis remains an effective way to separate proteins — so that the results of antibody-based immunodetection can be fairly unequivocal.

Click on the Sample Preparation & Gel Electrophoresis topics to read about the possible causes and remedies:

• No Bands or Gel Front
  
• Sample Doesn't Sink to the Bottom of the Well
  
• Sample Leaking Out of Well
  
• Bands Are Smeared Vertically
  
• Too Many Bands
  
• Gel Running Unusually Slowly
  
• Gel Running Unusually Fast
  
• Protein Bands Too Close Together (Not Completely Resolved)
  
• Leftmost and/or Rightmost Bands of the Gel are Distorted
  
• Bands Form Smiling Shapes

No Bands or Gel Front

Possible Cause	Remedy
Proteins have run off the gel	Decrease the amount of time the gel is run. Decrease the voltage. Ensure that the leads are in the correct orientation, as the electrophoresis leads to the power supply may be reversed, causing the gel to run upward. Increase the acrylamide percentage of the gel.
Not enough protein was loaded on the gel	Load more protein into each well. Ensure that the protein concentration of each sample is accurate.
Sample has diffused away from the well prior to applying power	Decrease the time between loading the first well of the gel and beginning electrophoresis. If necessary, run fewer gels and/or fewer lanes at once.
Not enough SDS in the sample	Without adequate SDS, the proteins are not negatively charged and will not travel through the cell. Ensure adequate SDS concentration.

Sample Doesn't Sink to the Bottom of the Well

Possible Cause	Remedy
Not enough glycerol in sample	Increase glycerol concentration.

Sample Leaking Out of Well

Possible Cause	Remedy
Wells damaged during comb removal	Remove gel comb carefully to avoid disruption of the wells. Rinse wells gently with electrophoresis buffer prior to sample loading to detect damaged wells.
Wells damaged during sample loading	Load sample carefully, ensuring that pipette tip does not touch the bottom or sides of the wells.

Bands are Smeared Vertically

Possible Cause	Remedy
Incomplete protein solubility blocks proteins from entering the gel	Ensure adequate sonication/homogenization and centrifugation during cell lysate preparation to remove particulate matter.
Protein degradation	Add/increase concentration of protease/phosphatase inhibitors during cell lysate preparation. Avoid repeated freeze/thaw cycles of cell lysate and Western blot samples.
Too much protein loaded Effect of loading too much protein on SDS-PAGE (rightmost lane)	Decrease the amount of protein loaded per well. Ensure accurate protein concentration of sample.
Gel run too fast	Increase gel run time.
Poor quality or old gel	Use a fresh gel.

Too Many Bands

Possible Cause	Remedy
Protein degradation	Add/increase concentration of protease/phosphatase inhibitors during cell lysate preparation. Avoid repeated freeze/thaw cycles of cell lysate and Western blot samples.
Protein aggregation	Ensure adequate DTT concentration, which reduces disulfide bonds. Heat Western blot samples in water bath prior to loading onto gel.

Gel Running Unusually Slowly

Possible Cause	Remedy
Buffers too concentrated	Dilute buffers.
Current too low	Increase voltage of gel apparatus.

Gel Running Unusually Fast

Possible Cause	Remedy
Buffers too dilute	Concentrate buffers.
Current too high	Decrease voltage of gel apparatus.

Protein Bands Too Close Together (Not Completely Resolved)

Possible Cause	Remedy
Insufficient run time	Run gel for longer period of time.
Incorrect gel pore size	Use a gradient gel (higher acrylamide percentage on bottom than top) that will adequately resolve a broader size range of proteins.

Leftmost and/or Rightmost Bands of the Gel are Distorted

Possible Cause	Remedy
Edge effects	Do not leave empty wells. Load wells not being used with a small amount of protein to prevent edge effects on neighboring lanes.

Bands Form Smiling Shapes

Possible Cause	Remedy
Excessive heat Gel run is “smiling.” This is caused by too high a voltage. Also, target bands are bleached, caused by using the detection reagent at too high of a concentration.	Run gel at a lower voltage for longer time. Use chilled buffers and run gel in a cold room or packed on ice.