Silver Staining of Proteins on Electroblotting Membranes and Intensification of Silver Staining of Proteins Separated by Polyacrylamide Gel Electrophoresis

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 mem...

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Bibliographic Details
Published in:Analytical biochemistry 2002-05, Vol.304 (1), p.33-41
Main Authors: Sørensen, Birgitte Kjær, Højrup, Peter, Østergård, Erik, Jørgensen, Charlotte Sværke, Enghild, Jan, Ryder, Lisa Rebekka, Houen, Gunnar
Format: Article
Language:English
Subjects:
Cell Line
Collodion
electrophoresis
Electrophoresis, Gel, Two-Dimensional - methods
Electrophoresis, Polyacrylamide Gel - methods
Endothelium - chemistry
Gallyas' stain
Humans
Mass Spectrometry
Membranes, Artificial
Polyvinyls
proteins
Proteins - isolation & purification
Silver
silver intensification
Staining and Labeling - methods
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Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.2001.5604