Título:
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Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis.
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Autores:
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Birgitte Kjær Sørensen, Autor ;
Peter Højrup, Autor ;
Erik Østergård, Autor ;
Charlotte Sværke Jørgensen, Autor ;
Jan Enghild, Autor ;
Lisa Rebekka Ryder, Autor ;
Gunnar Houen, Autor
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Tipo de documento:
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documento electrónico
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Fecha de publicación:
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2002
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ISBN/ISSN/DL:
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68036
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Nota general:
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En: Analytical Biochemistry.-- 2002, 304:33–41
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Langues:
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Inglés
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Clasificación:
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ELECTROFORESIS
ESPECTROMETRÍA DE MASA
PROTEÍNAS
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Resumen:
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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.
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