A luminescent europium complex for the sensitive detection of proteins and nucleic acids immobilized on membrane supports

Abstract

Certain metal complexes selectively interact with proteins immobilized on solid-phase membrane supports to form brightly colored products. Detecting the absorbance of calorimetric stains is limited by the molar extinction coefficient of the product, however. Development of light-emitting complexes should improve detection sensitivity, but fluorescent labels described to date modify free amino, carboxyl, or sulfhydryl groups often rendering proteins unsuitable for further analysis. Bathophenanthroline disulfonate (BPSA) forms a luminescent europium (Eu) complex that reversibly binds to proteins and nucleic acids. Analysis of charge-fractionated carrier ampholytes and synthetic polymers of different L-amino acids indicates that protein binding is chiefly through protonated α-and ε-amino side chains. Proteins or nucleic acids immobilized to a nitrocellulose or polyvinyl difluoride membrane by electroblotting, dot-blotting, or vacuum slot-blotting are incubated with the lanthanide complex at acidic pH. Membranes are rinsed, illuminated with UV light and the phosphorescence of BPSA-Eu is measured at 590 to 615 nm using a CCD camera or spectrofluorimeter. The linear dynamic range of the stain is 476- and 48-fold for protein and DNA, respectively. A strong chelating agent such as ethylenediaminetetraacetic acid combined with a shift to basic pH (pH 8-10) elutes BPSA-Eu from the membrane. The reversible nature of the protein staining procedure allows for subsequent biochemical analyses, such as immunoblotting, lectin staining, and mass spectrometry.