Western Blotting 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

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