Detection of Immobilized Proteins on Nitrocellulose Membranes Using a Biotinylation-Dependent System

✍ Scribed by Rüdiger Graf; Peter Friedl

• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 210 KB
• Volume: 273
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.4237

✦ Synopsis

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.