Charge-coupled device camera-based detection of fluorescence-labeled proteins immobilized on nitrocellulose membranes.
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.
Graf
R
R
Institut für Biochemie, Technische Universität, Darmstadt, Germany.
Friedl
P
P
D019921:Biotinylation; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000295:instrumentation; D005562:Formazans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation; D007074:Immunoglobulin G / Q000032:analysis; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D008165:Luminol; D009195:Peroxidase; D011506:Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity; D019809:Streptavidin
D005456:Fluorescent Dyes; D005562:Formazans; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D011506:Proteins; D008165:Luminol; D003101:Collodion; D019809:Streptavidin; D009195:Peroxidase; D016650:Fluorescein-5-isothiocyanate