A simple, two-color fluorescence detection method for membrane blotting analysis using alkaline phosphatase and horseradish peroxidase.

We have developed a one-step, two-color fluorescence detection method using simultaneously two fluorogenic substrates for both Southern and Western blots on nylon membranes. For this enzyme-mediated reporter system, a mixture of (i) 3-hydroxy-N-2'-biphenyl-2-naphthalenecarboxamide phosphate ester (HNPP), a substrate for alkaline phosphatase and (ii) N-(4-amino-5-methoxy-2-methylphenyl)benzamide (AMMB), a fluorogenic substrate for horseradish peroxidase was used. The reaction with these substrates produces blue (HNPP) and yellow (AMMB) fluorescent signals under ultraviolet light (302 nm). Therefore, this simple method allows the simultaneous visualization of two different targets on a single nylon membrane, e.g. nucleic acids or proteins.