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 visua lized by streptavidin-peroxidase-based detection systems either by depositi on of a colored formazan dye or by enhanced chemiluminescence (ECL), the la tter being 10-fold more sensitive. The sensitivity of the staining procedur e is dramatically improved by the inclusion of the reporter deposit techniq ue into the staining procedure: the initially bound peroxidase generates ph enolic radicals from biotinyltyramide, enhancing the number of biotinyl res idues in the vicinity of the first biotinylation site. Thus the detection l imit is lowered to 1 pg of protein with the ECL detection. The new method i s compared with silver stain and immunochemical staining in Western blots a nd furthermore its suitability is demonstrated for 2-D gel electrophoresis. (C) 1999 Academic Press.