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Sample Preparation and Gel Electrophoresis

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Related Resources: Brochures | Application Notes
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 and Gel Electrophoresis topics to read about the possible causes and remedies:

No Bands or Gel Front

Possible CauseRemedy
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
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.
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Sample Doesn’t Sink to the Bottom of the Well

Possible CauseRemedy
Not enough glycerol in sample
  • Increase glycerol concentration.
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Sample Leaking Out of Well

Possible CauseRemedy
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.
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Bands Are Smeared Vertically

Possible CauseRemedy
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.
  • Explore inhibitor cocktail options
  • 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)
Effect of loading too much protein on SDS-PAGE (rightmost lane)
 
Gel run too fast
  • Increase gel run time.
Poor quality or old gel
  • Use a fresh gel.
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Too Many Bands

Possible CauseRemedy
Protein degradation
  • Add/increase concentration of protease/phosphatase inhibitors during cell lysate preparation.
  • Explore inhibitor cocktail options
  • 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.
  • Order OmniPur® DTT
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Gel Running Unusually Slowly

Possible CauseRemedy
Buffers too concentrated
  • Dilute buffers.
Current too low
  • Increase voltage of gel apparatus.
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Gel Running Unusually Fast

Possible CauseRemedy
Buffers too dilute
  • Concentrate buffers.
Current too high
  • Decrease voltage of gel apparatus.
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Protein Bands Too Close Together (Not Completely Resolved)

Possible CauseRemedy
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.
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Leftmost and/or Rightmost Bands of the Gel are Distorted

Possible CauseRemedy
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.
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Bands Form Smiling Shapes

Possible CauseRemedy
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.
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.
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