The appearance of white bands ("ghost bands") on blots is usually caused by an intense localized signal, that completely exhausts the ECL reaction with a quick burst of light. Therefore, there is no light produced during development, and a white band occurs. This can also happen with prolonged incubation.

How to solve this issue?

  • Use less of the primary and secondary antibodies, e.g. dilute them further
  • Decrease protein load/well (here we explain why it is good to measure protein concentration in your samples, and how to do it in the presence of SDS, urea or detergent)
  • Use a high-titer secondary antibody, for example Agrisera's exceptional Goat anti-Rabbit IgG, HRP-conjugated (AS09 602, Agrisera). This will also help avoid this problem, as it can be used in dilutions of 1: 10 000 and higher. Using a secondary antibody at a high dilution will also help minimize the overall background signal.  
 Ghost bands on the blot
Example of a Western blot with the "ghost bands" appearing during chemiluminescent detection.

 Technical blog