Silver Staining of Proteins on Electroblotting Membranes and Intensification of Silver Staining of Proteins Separated by Polyacrylamide Gel Electrophoresis Birgitte Kjær Sørensen,* Peter Højrup,† Erik Østergård,* Charlotte Sværke Jørgensen,* Jan Enghild,‡ Lisa Rebekka Ryder,* and Gunnar Houen* ,1 *Department of Research and Development, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark; †Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark; and ‡Department of Molecular and Structural Biology, University of Aarhus, Gustav Wieds vej 10C, DK-8000 Aarhus C, Denmark Received September 7, 2001; published online April 1, 2002 A fast and convenient method for silver staining of proteins on electroblotting membranes was developed based on Gallyas’ histochemical intensifier and ap- plied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electro- blotted 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. Further- more, an intensifying method for proteins in silver- stained SDS-PAGE gels was developed based on Gall- yas’ histochemical intensifier. This method was applied to proteins separated by one- and two-dimen- sional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensifica- tion was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensi- tive 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. Pro- teins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the inten- sification procedure was fully compatible with mass spectrometry. © 2002 Elsevier Science (USA) Key Words: proteins; electrophoresis; silver intensi- fication; mass spectrometry; Gallyas’ stain. Silver staining, introduced in 1979 by Switzer et al. (1), has provided a breakthrough for sensitive protein detection. Numerous silver staining protocols have been described, each with different advantages regard- ing speed, sensitivity, cost, and compatibility with other analytical methods, notably MS, and the detec- tion limit is about 100 times lower than that of Coo- massie brilliant blue staining (2– 6). The basic mecha- nisms of silver staining in gels are relatively well understood, involving binding of silver ions to the pro- teins followed by reduction to free silver, and in addi- tion, various methods for sensitization and enhance- ment have been developed (7). The different protocols can be divided into alkaline and acidic methods, the alkaline of which are most sensitive but require longer procedures, whereas the acidic protocols are faster but less sensitive. The sensitivity of silver staining is in the same range as mass spectrometry, making it desirable to avoid introduction of chemical modifications in the proteins during staining. Many silver-staining proce- dures use glutaraldehyde in the sensitization step, which may interfere with protein identification due to the introduction of cross-links in proteins (8). In contrast to the wealth of protocols for silver stain- ing of proteins in gels only few protocols for silver staining of proteins on electroblotting membranes have been described (9 –14). Moreover, most of these meth- ods are either laborious or have a low sensitivity. In this report, we describe a fast, convenient, and sensi- tive method for silver staining of proteins on electro- blotting membranes based on Gallyas’ histochemical developer (15–17). Moreover, we describe the use of this developer for intensifying silver images in poly- acrylamide gels in combination with a number of dif- 1 To whom correspondence should be addressed. Fax: +45 32 68 31 49. E-mail: gh@ssi.dk. 0003-2697/02 $35.00 33 © 2002 Elsevier Science (USA) All rights reserved. Analytical Biochemistry 304, 33– 41 (2002) doi:10.1006/abio.2001.5604, available online at http://www.idealibrary.com on