Western Blot 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
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
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



Western Blot Troubleshoot: Unusual or Unexpected Bands Western Blot Troubleshoot: Faint Bands or Weak Signal