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. 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.
Lim
M J
MJ
Boston University, Biological Sciences Department, Massachusetts 02215, USA.
Patton
W F
WF
Lopez
M F
MF
Spofford
K H
KH
Shojaee
N
N
Shepro
D
D
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D003101:Collodion / Q000737:chemistry; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D004492:Edetic Acid / Q000737:chemistry; D004586:Electrophoresis / Q000379:methods; D005063:Europium / Q000737:chemistry* / Q000378:metabolism; D005069:Evaluation Studies as Topic; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008163:Luminescent Measurements; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D009696:Nucleic Acids / Q000032:analysis* / Q000737:chemistry; D010618:Phenanthrolines / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012313:RNA / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
D008567:Membranes, Artificial; D009696:Nucleic Acids; D010618:Phenanthrolines; D011506:Proteins; C017049:bathophenanthroline disulfonic acid; D005063:Europium; D012313:RNA; D003101:Collodion; D004247:DNA; D004492:Edetic Acid