Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals. It is compatible with downstream processing, such as mass spectrometry analysis after protein digestion. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 d after the end of the electrophoretic separation. Once completed, the stain is stable for several weeks.
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Rabilloud, T. Mechanisms of protein silver staining in polyacrylamide gels: a 10-year synthesis. Electrophoresis 11, 785–794 (1990).
Merril, C.R. et al. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211, 1437–1438 (1981).
Schagger, H. & von Jagow, G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379 (1987).
Wiltfang, J. et al. A new multiphasic buffer system for sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100,000–1000, and their detection with picomolar sensitivity. Electrophoresis 12, 352–366 (1991).
Tastet, C. et al. A versatile electrophoresis system for the analysis of high- and low-molecular weight proteins. Electrophoresis 24, 1787–1794 (2003).
Hochstrasser, D.F. & Merril, C.R. 'Catalysts' for polyacrylamide gel polymerization and detection of proteins by silver staining. Appl. Theor. Electrophor. 1, 35–40 (1988).
Eschenbruch, M. & Bürk, R.R. Experimentally improved reliability of ultrasensitive silver staining of protein in polyacrylamide gels. Anal. Biochem. 125, 96–99 (1982).
Chevallet, M. et al. Improved mass spectrometry compatibility is afforded by ammoniacal silver staining. Proteomics 6, 2350–2354 (2006).
Blum, H. et al. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93–99 (1987).
Rabilloud, T. A comparison between low background silver diammine and silver nitrate protein stains. Electrophoresis 13, 429–439 (1992).
Richert, S. et al. About the mechanism of interference of silver staining with peptide mass spectrometry. Proteomics 4, 909–916 (2004).
Sinha, P. et al. A new silver staining apparatus for MALDI/TOF analysis of proteins after two-dimensional electrophoresis. Proteomics 1, 835–840 (2001).
Mold, D.E. et al. Silver staining of histones in Triton-acid-urea gels. Anal. Biochem. 135, 44–47 (1983).
Rabilloud, T. et al. Two-dimensional electrophoresis of human placental mitochondria and protein ientification by mass spectrometry: Toward a human mitochondrial proteome. Electrophoresis 19, 1006–1014 (1998).
Gharahdaghi, F. et al. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 20, 601–605 (1999).
Neuhoff, V. et al. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9, 255–262 (1988).
The authors declare no competing financial interests.
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Chevallet, M., Luche, S. & Rabilloud, T. Silver staining of proteins in polyacrylamide gels. Nat Protoc 1, 1852–1858 (2006). https://doi.org/10.1038/nprot.2006.288
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