A LUMINESCENT EUROPIUM COMPLEX FOR THE SENSITIVE DETECTION OF PROTEINS AND NUCLEIC-ACIDS IMMOBILIZED ON MEMBRANE SUPPORTS

Certain metal complexes selectively interact with proteins immobilized on solid-phase membrane supports to form brightly colored products. D etecting the absorbance of colorimetric stains is limited by the molar extinction coefficient of the product, however. Development of light- emitting complexes should improve detection sensitivity, but fluoresce nt labels described to date modify free amino, carboxyl, or sulfhydryl groups often rendering proteins unsuitable for further analysis. Bath ophenanthroline disulfonate (BPSA) forms a luminescent europium (Eu) c omplex that reversibly binds to proteins and nucleic acids. Analysis o f charge-fractionated carrier ampholytes and synthetic polymers of dif ferent L-amino acids indicates that protein binding is chiefly through protonated gamma-and epsilon-amino side chains. Proteins or nucleic a cids immobilized to a nitrocellulose or polyvinyl difluoride membrane by electroblotting, dot-blotting, or vacuum slot-blotting are incubate d with the lanthanide complex at acidic pH. Membranes are rinsed, illu minated with UV light and the phosphorescence of BPSA-Eu is measured a t 590 to 615 nm using a CCD camera or spectrofluorimeter. The linear d ynamic range of the stain is 476- and 48-fold for protein and DNA, res pectively. 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, le ctin staining, and mass spectrometry. (C) 1997 Academic Press.