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

✍ Scribed by Birgitte Kjær Sørensen; Peter Højrup; Erik Østergård; Charlotte Sværke Jørgensen; Jan Enghild; Lisa Rebekka Ryder; Gunnar Houen


Publisher
Elsevier Science
Year
2002
Tongue
English
Weight
323 KB
Volume
304
Category
Article
ISSN
0003-2697

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✦ Synopsis


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 silverstained 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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