Western Blotting Troubleshooting: No Bands

Source of Problem	Possible Cause	Action/Solution
Antibody related	Inappropriate secondary antibody used	Retrace steps to check compatibility between primary and secondary antibodies Reprobe with correct secondary or strip blot and reprobe if necessary Repeat experiment with the correct antibody combination
	Wrong concentration of antibody or low affinity to the target protein	Increase the antibody concentration 2-4 fold higher than initially recommended Increase length of incubation Test/optimize antibody on dot blots Try another antibody
	Antibody not suitable for Western blotting	Check datasheet for recommended conditions Test/optimize antibody on dot blots Try alternate antibody
	Antibody has lost activity due to long term or improper storage	Test on a dot blot at several concentrations Use fresh aliquot of antibody that has been stored at -20°C or below
Antigen related	Antigen not expressed in the source material	Use another source of target protein
Antigen related	Not enough antigen loaded on the gel	Check concentration of sample Increase the amount of source material Immunoprecipitate, fractionate, or concentrate the sample
Technique related	Transfer did not work properly	Confirm protein transfer by staining the membrane with Ponceau S and/or the gel with Coomassie dye. Note how well any prestained molecular weight markers have transferred onto the blot Optimize/check transfer conditions and set up (especially orientation to electrodes) Repeat using two membranes in case protein has transferred through the first
Technique related	Washes are too stringent	Use fewer washers Reduce washing time
Buffer related	Blocking agent is interfering with signal	Try lower concentration Try alternate blocking agent
	Buffers may contain sodium azide which inactivates HRP	Use azide free buffers
	Peroxide may be inactive reducing activity of peroxidase	Add fresh peroxide to substrate buffer
	ECL detection reagents have been contaminated	Use fresh detection reagents

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