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

Anal Biochem. 1997 Feb 15;245(2):184-95. doi: 10.1006/abio.1996.9961.

Abstract

Certain metal complexes selectively interact with proteins immobilized on solid-phase membrane supports to form brightly colored products. Detecting 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 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 alpha- and epsilon-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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Biochemistry / methods*
  • Collodion / chemistry
  • DNA / analysis
  • DNA / chemistry
  • DNA / metabolism
  • Edetic Acid / chemistry
  • Electrophoresis / methods
  • Europium / chemistry*
  • Europium / metabolism
  • Evaluation Studies as Topic
  • Hydrogen-Ion Concentration
  • Immunoblotting
  • Luminescent Measurements*
  • Mass Spectrometry
  • Membranes, Artificial
  • Nucleic Acids / analysis*
  • Nucleic Acids / chemistry
  • Phenanthrolines / chemistry
  • Phenanthrolines / metabolism
  • Proteins / analysis*
  • Proteins / chemistry
  • RNA / analysis
  • RNA / chemistry
  • RNA / metabolism
  • Sensitivity and Specificity
  • Staining and Labeling / methods

Substances

  • Membranes, Artificial
  • Nucleic Acids
  • Phenanthrolines
  • Proteins
  • bathophenanthroline disulfonic acid
  • Europium
  • RNA
  • Collodion
  • DNA
  • Edetic Acid