What is a reason for so called "ghost bands" to appear on blots?

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.