Western Blot Troubleshooting: Faint Bands or Weak Signal

Source of Problem Possible Cause Action/ Solution
Antibody related

Primary or secondary antibody concentrations were too low

  • Repeat using higher concentration of antibody
  • Optimize antibody concentration with dot blots
Low antigen-antibody binding affinity
  • Reduce the number of wash steps to minimum
  • Increase the antibody concentration 2-4 fold higher than the recommended starting dilution
Antigen related Insufficient sample loaded on the gel
  • Check concentration of sample
  • Increase the amount of source material 
  • Immunoprecipitate, fractionate, or concentrate sample
Blot has been stripped and reporbed
  • Redo blot since antigen may have been stripped off or damaged by stripping process
Technique related Low transfer efficiency
  • Confirm transfer with Ponceau S staining
  • Optimize transfer conditions for target protein size
  • Optimize transfer buffers for methanol and SDS concentrations
Film exposure time (ECL detection) was too short
  • Test different exposure times
Buffer related Non-fat dry milk may mask some antigens
  • Decrease % of milk in the blocking and antibody solutions
  • Try alternate blocking solution



Western Blot Troubleshoot: No Bands Western Blot Troubleshoot: High Background Signal on the Blot