Several variables may account for this:
a.This may be due to inadequate transfer of proteins during electroblotting. Try staining the membrane with India Ink or Ponceau-S immediately after the transfer. This is an effective and non-interfering method to verify proper protein transfer. Consult our recommended Western blotting protocols to optimize these electrophoresis and transfer conditions.
b. The detection enzyme may be inactivated. Sodium azide inactivates horseradish peroxidase (HRP) irreversibly, so do not include sodium azide in any HRP-labeled reagents. Bacterial contamination also diminishes HRP activity. HRP conjugates should be kept bacteria-free, handled with sterile technique and stored under recommended conditions.
c.The polyvinyl wrap from certain sources may quench the signal. We suggest repeating the incubation with chemiluminescence reagents and placing the blot between two pieces of write-on acetate transparency film, and then expose the film.
d.The expression of the protein of interest may be very low. Sensitivity may be increased by performing an immunoprecipitation prior to the Western blot.
e.In cases of very weak Ag expression, eliminating Tween during primary Ab incubation may improve Ab binding.
f.To better facilitate Ag-Ab interaction you could incubate primary Ab at 37°C with gentle incubation and further incubate it overnight at 4°C.