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Sample Preparation & Gel Electrophoresis

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

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

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.

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.