Western Blot Troubleshooting: High Background Signal on the Blot

Source of Problem Possible Cause Action/ Solution
Antibody related
Concentration of primary and/or secondary antibody too high
  • Reduce/optimize the antibody concentrations 
  • Reduce/optimize incubation times
  • Use affinity purified antibodies
Primary and/or secondary binds to the blocking agent
  • Use alternate blocking solution
Antibody has reduced activity due to long term or improper storage
  • Use a fresh aliquot of antibody that has been stored at -20°C or below
Phospho-specific antibody has reacted with casein (a phosphoprotein) in nonfat milk blocking agent
Secondary antibody is binding to blocking agent
  • Try another blocking agent
  • Reduce protein concentration of blocking agent
  • Test secondary antibody alone on blot
  • Try alternate form of secondary, such as F(ab’)2
Antigen related
Non-specific interaction with genomic DNA in samples
  • Sonicate the lysates
  • Add DNAse to lysis buffer
Technique related
Membrane blocking is not sufficiant
  • Confirm concentration matches that recommended in the protocol
  • Try other blocking agents
  • Try alternate incubation temperatures, including room temperature
Membrane has dried out
  • Repeat procedure taking care that the blot does not dry out during any step by using sufficient volumes and agitation throughout
PVDF membrane has higher background
  • Try nitrocellulose instead
Inadequate washing between incubations
  • Increase the length of washing steps
  • Use a larger volume of wash buffer
Incubation temperature too high
  • Try alternate incubation temperatures such as 4°C
Film over-exposed or blot developed for too long
  • Wait 5-10 minutes and then re-expose blot to film
  • Reduce exposure and/or development times
Buffer related
Insufficient concentration of detergent in the buffers
  • Use TBS containing >0 .1% Tween® 20
  • Try stronger detergent, such as NP-40
Transfer, incubation, or blocking solutions are contaminated
  • Use fresh buffers

Western Blot Troubleshoot: Faint Bands or Weak Signal Western Blot Troubleshoot: Patchy or Uneven Spots on the Blot