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Charge‐coupled device camera‐based detection of fluorescence‐labeled proteins immobilized on nitrocellulose membranes
Author(s) -
Graf Rüdiger,
Friedl Peter
Publication year - 2001
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683()22:5<890::aid-elps890>3.0.co;2-1
Subject(s) - nitrocellulose , streptavidin , chemistry , membrane , peroxidase , collodion , fluorescence , biotinylation , biotin , chromatography , biochemistry , enzyme , physics , quantum mechanics
Proteins dotted on nitrocellulose membranes are biotinylated by reaction with a biotinyl‐succinimide ester. The resulting biotinyl residues serve as specific binding sites for a subsequent streptavidin‐based detection system. Using streptavidin‐peroxidase, the proteins are visualized either by deposition of a colored formazan dye or by enhanced chemiluminescence the latter being twofold more sensitive. Alternatively, streptavidin‐fluorescein isothiocyanate (FITC) is substituted for the peroxidase conjugate as tool for protein staining. The sensitivity of both staining variants is dramatically improved by the inclusion of the reporter deposition technique. The fluorescence‐labeled proteins are visualized on a visible blue light emitting illuminator preventing the bothering effect of photobleaching. In combination with a charge‐coupled device (CCD) camera‐based image analyzing system the established stain with streptavidin‐FITC detects about 10 pg of protein dot blotted on nitrocellulose membranes.