Western Blotting 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
Antibody related	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
Antigen related	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
Technique related	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

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