Quantitative western blot analysis and spot immunodetection using time-resolved fluorometry.
We describe a new method of staining and quantification of proteins blotted or spotted on nitrocellulose. Blotted or spotted proteins are first reacted with specific antibodies followed by reaction with biotinylated secondary antibodies. The immunocomplex is then reacted with a streptavidin-based macromolecular complex labeled with the fluorescent europium chelate of 4,7-bis(chlorosulfophenyl) 1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). The fluorescent spots or bands can then be assessed by visual inspection under UV illumination, by instant photography or quantified by scanning with a time-resolved fluorometer. The method does not involve enzyme detection, is simple, sensitive and gives sharp bands which remain fluorescent for long periods of time (months to years).
Department of Clinical Biochemistry, Toronto Western Hospital, Ontario, Canada.
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Diamandis
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Christopoulos
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Bean
D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002272:Carcinoembryonic Antigen / Q000032:analysis; D002614:Chelating Agents; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D005063:Europium; D005293:Ferritins / Q000032:analysis; D005456:Fluorescent Dyes; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D010618:Phenanthrolines; D011506:Proteins / Q000032:analysis*; D000509:alpha-Fetoproteins / Q000032:analysis
D002272:Carcinoembryonic Antigen; D002614:Chelating Agents; D005456:Fluorescent Dyes; D010618:Phenanthrolines; D011506:Proteins; D000509:alpha-Fetoproteins; C054206:4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid; D005063:Europium; D005293:Ferritins