Detection of immobilized proteins on nitrocellulose membranes using a biotinylation-dependent system.
Proteins are biotinylated after immobilization on nitrocellulose sheets by reaction with a biotinyl-succinimide ester. The biotinyl residues are visualized by streptavidin-peroxidase-based detection systems either by deposition of a colored formazan dye or by enhanced chemiluminescence (ECL), the latter being 10-fold more sensitive. The sensitivity of the staining procedure is dramatically improved by the inclusion of the reporter deposit technique into the staining procedure: the initially bound peroxidase generates phenolic radicals from biotinyltyramide, enhancing the number of biotinyl residues in the vicinity of the first biotinylation site. Thus the detection limit is lowered to 1 pg of protein with the ECL detection. The new method is compared with silver stain and immunochemical staining in Western blots and furthermore its suitability is demonstrated for 2-D gel electrophoresis.
Graf
R
R
Institut für Biochemie, Technische Universität Darmstadt, Petersenstrasse 22, Darmstadt, D-64287, Germany.
Friedl
P
P
D001710:Biotin / Q000031:analogs & derivatives; D019921:Biotinylation; D015153:Blotting, Western; D003101:Collodion; D015180:Electrophoresis, Gel, Two-Dimensional; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D008567:Membranes, Artificial; D010544:Peroxidases; D011506:Proteins / Q000032:analysis*; D012834:Silver; D013194:Staining and Labeling / Q000379:methods*; D019809:Streptavidin; D014439:Tyramine / Q000031:analogs & derivatives
D007202:Indicators and Reagents; D008567:Membranes, Artificial; D011506:Proteins; C111551:biotinyltyramide; D012834:Silver; D001710:Biotin; D003101:Collodion; D019809:Streptavidin; D010544:Peroxidases; D014439:Tyramine