Nonuniform variation in band pattern with luminol/horseradish peroxidase western blotting.

When optimized, the peroxidase-catalyzed oxidation of luminol can provide a useful, sensitive detection system for Western blotting. However, while the luminescence from intense bands appears rapidly, faint bands require at least 30 min after removal of the membrane from reaction buffer for maximum luminescence to develop. This can result in the detection of a variant band pattern if films are exposed to the blotted membrane too soon after reaction, while exposure later after reaction can result in the preferential detection of faint bands. As a consequence, in order to detect a range of bands similar to that seen using autoradiographic or chromogenic systems, it is necessary to determine the correct time after the initiation of the luminol reaction for film exposure. These effects are due to enhancement of luminescence as a result of the peroxidase-immunoglobulin conjugate binding to a solid phase.