Western Blot Troubleshooting: Unusual or Unexpected Bands

Difference seen Possible Cause Action/ Solution
Band(s) at lower molecular weight than expected
  • Target protein has been cleaved or digested
  • Splice variants exist
  • Another protein bearing the same/similar epitope has been detected by antibody
  • Use a fresh sample which has been kept on ice
  • Add fresh protease inhibitors to the lysis buffer
  • Try alternate antibody
Band(s) at slightly higher molecular weight than expected, and may be blurred
Protein may be glycosylated or otherwise modified at one or more amino acid residues
  • Use enzymes to remove suspected modification returning molecular weight closer to expected
  • Check amino acid sequence and literature
Band(s) at significantly higher molecular weight than expected
Dimers, multimers, or protein-protein interactions may be occuring because samples have not been fully reduced and denatured.
  • Add fresh DTT or bME to samples and reheat before repeating experiment
  • Prepare new samples with fresh loading buffer
Multiple bands at various molecular weights
Primary antibody concentration may be too high, or there
is a cross-reactivity with similar epitopes on other proteins
  • Use an affinity-purified primary antibody
  • Optimize primary antibody concentration
  • Try another antibody
  • Check antibody specificity with blocking peptide
Secondary antibody concentration is too high leading to non-specific binding
  • Decrease/optimize the concentration of the secondary antibody
  • Use an affinity-purified secondary antibody
  • Repeat immunodetection with secondary antibody alone to check for non-specific binding
Protein exists in several different isoforms
  • Check literature
Bands are blurry Gel was run at too high a voltage
  • Repeat gel at lower voltage
Incorrect running buffer composition
  • Prepare fresh running buffer
Trapped air bubble present during transfer
  • Carefully remove air bubbles between the gel and the membrane before protein transfer
Bands are smile shaped, not flat
Running conditions were too fast
so gel became over heated
  • Check and optimize gel electrophoresis conditions
  • Run gel at 4°C
White (negative) bands on the film when using ECL detection
Too much protein has been loaded
  • Load less sample
  • Repeat with dilution series of sample
Antibody concentration is too high
  • Reduce/optimize the antibody

Chapter 6: Western Blot Troubleshooting Western Blot Troubleshoot: No Bands