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AAT Bioquest

What are the possible causes for nonspecific or diffuse bands for my western blot experiment?

Posted September 13, 2024


Answer

Possible cause 

Solution 

Excessive amounts of protein loaded on gel

Load less sample on the gel

High antibody concentration 

Use a lower concentration of antibodies, particularly for primary antibodies 

Chemiluminescent substrate emits a very strong signal

  • Use a less concentrated substrate
  • Reduce duration of membrane incubation with substrate
  • Expose blot to film for a shorter duration
  • Use a lower antibody concentration, particularly AP- and HRP- conjugated antibodies
  • Remove substrate completely after incubation is complete
Additional resources

Western Blot: Technique, Theory, and Trouble Shooting

Western Blotting Assays

iFluor® 488 goat anti-mouse IgG (H+L) *Cross Adsorbed*