Western Blotting 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	Use alternate blocking solution- BSA or Block Ace (Bio-Rad BUF029)
	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
Buffer related	Transfer, incubation, or blocking solutions are contaminated	Use fresh buffers

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