Western Blot Troubleshooting: Patchy or Uneven Spots on the Blot

Source of Problem Possible Cause Action/ Solution
Antibody related
HRP-linked secondary antibody has aggregated
  • Spin secondary antibody and filter to remove aggregates
Technique related
Not enough solution used during incubation and/or washing
  • Make sure the membrane is fully immersed and agitated throughout incubations
Trapped air bubbles
  • Carefully remove air bubbles between the gel and the membrane before protein transfer
Uneven agitation during incubation
  • Use a shaker for all incubations
Buffer related Antibodies are binding to the blocking agent
  • Filter the blocking agent
  • Try another blocking agent

Western Blot Troubleshoot: High Background Signal on the Blot Introduction to Western Blotting