title: Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis.
abstract: A fast and convenient method for silver staining of proteins on electroblotting membranes was developed based on Gallyas' histochemical intensifier and applied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electroblotted to polyvinyl difluoride membranes. The method allowed detection of proteins on membranes with a sensitivity equal to the sensitivity of the most sensitive silver-staining protocols for electrophoresis gels. Also, the method was compatible with preceding immunostaining on the same membrane. Furthermore, an intensifying method for proteins in silver-stained SDS-PAGE gels was developed based on Gallyas' histochemical intensifier. This method was applied to proteins separated by one- and two-dimensional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensification was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensitive alkaline protocols a significant increase in signal to noise ratio was obtained. In particular, negatively stained or invisible proteins on the silver-stained gels were found to be visualized by the Gallyas stain. Proteins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the intensification procedure was fully compatible with mass spectrometry.
authors.affiliation: Department of Research and Development, Statens Serum Institut, Artillerivej 5, DK-2300, Copenhagen S, Denmark.
authors.forename: Birgitte Kjaer
authors.initials: BK
authors.lastname: Sørensen
authors.forename: Peter
authors.initials: P
authors.lastname: Højrup
authors.forename: Erik
authors.initials: E
authors.lastname: Østergård
authors.forename: Charlotte Svaerke
authors.initials: CS
authors.lastname: Jørgensen
authors.forename: Jan
authors.initials: J
authors.lastname: Enghild
authors.forename: Lisa Rebekka
authors.initials: LR
authors.lastname: Ryder
authors.forename: Gunnar
authors.initials: G
authors.lastname: Houen
mesh_terms: D002460:Cell Line; D003101:Collodion; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004727:Endothelium / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins / Q000302:isolation & purification*; D012834:Silver; D013194:Staining and Labeling / Q000379:methods*; D005465:Fluorocarbon Polymers
chemical_list: D003101:Collodion; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; D012834:Silver; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers