Inexpensive chemifluorescent detection of antibody-alkaline phosphatase conjugates on Western blots using 4-methylumbelliferyl phosphate.

Nonradioactive methods for the detection of immobilized protein on Western blots are standard laboratory practice. This is in contrast to nucleic acid blotting, which still commonly involves the use of radioactive probes. Although the nonradioactive detection of antibody (Ab)1 complexes in Western blotting can employ direct detection of Xuorochrome–Ab conjugates, the majority of systems detect products produced by the activity of the enzyme conjugates alkaline phosphatase (AP) and horseradish peroxidase (HRP) [1,2]. The current study arose from numerous reports on the detection of phosphatase activity in native protein gels and in solution using the substrate 4-methylumbelliferyl phosphate (4-MUP). Although the use of 4-MUP for the detection of AP and AP–Ab conjugates in the probing of immobilized DNA [3,4] and virus [5] has been documented, we were surprised to Wnd no references to its use as a reagent for the detection of bound target molecules on Western blots. Therefore, we tested whether this substrate could be used to detect AP activity immobilized as an Ab conjugate on Western blots. Relative sensitivity of the detection of AP–Ab conjugates using 4-MUP was investigated in comparison with other chemiXuorescent kits designed for the detection of AP–Ab conjugates (commer-