Silver staining, two-dimensional gel

Figure 5.18 Silver-stained two-dimensional gel of the proteins extracted from the yeast S. cerevisiae. From Poutanen, M., Salusjarvi, L., Ruohonen, L., Penttila, M. and KaLkM-nen, N., Rapid Commun. Mass Spectrom., 15, 1685-1692, Copyright 2001. John WUey Sons Limited. Reproduced with permission.

Gorg, A., Postel, W., Weser, J., Gunther, S., Strahler, J.R., Hanash, S.M. and Somerlot, L. (1987) Elimination of point streaking on silver stained two-dimensional gels by addition of iodoacetamide to the equilibration buffer. Electrophoresis 8, 122-124. 

Widespread use of mass spectrometry to analyze large numbers of spots from two-dimensional gels has also brought out another apparent shortcoming of 2DE. Articles on proteome-wide analyses of Saccharomyces cerevisiae point to a growing awareness in the literature and community of another shortcoming of 2DE, namely that no spots with low abundance can be identified from silver stained two-dimensional gels. °-"... 

Fig. 6.15. Silver-stained two-dimensional gel of platelet proteins (courtesy of Dr. Sammons).

Figure 21.10 presents such an image that resembles a silver-stained two-dimensional gel. The image consists of a set of spots, distributed across the surface. The positions of these spots are highly reproducible from cell to cell, with typical precision in spot position being on the order of the size of the spot itself. 

Figure 8. Details of high-resolution silver stained two-dimensional polyacrylamide gels of the blood lymphocytes obtained from healthy donors. A B lymphocytes (CD19 ), B T lymphocytes (CD8 ), C T lymphocytes (CD4 ). The spots corresponding to vimentin. tubulin, desmin or cytokeratin are in the frame. First dimension immobilized 3 to 10 pH gradient, second dimension 9 to 16% polyacrylamide gel electrophoresis [adapted from (Vuadens et al., 2002), with permission of the publisher].

Figure 4. Separation and detection of ECPs by two dimensional gel electrophoresis. A Silver stained. B Immunoblot with conventional procedure day 112 antisera. C Immuunoblot with cascade procedure day 112 antisera. D Immunoblot with passive immunization procedure day 112 antisera. Exposure time was 24 hours. Reproduced with permission from Ref. 24. Copyright 1989 The Humana Press Inc.

A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling. 

Hanash, S.M., Tuhergen, D.G., Heyn, R.M., Neel, J.V., Sandy, L., Stevens, G.S., Rosenhlum, B.B., and Krzesicki, R.F., 1982, Two-dimensional gel electrophoresis of cell proteins in childhood leukemia, with silver staining a preliminary report. Clin. Chem. 28 1026-1030. 

Additional biochemical characterization of in vitro disassembly products can be achieved through two-dimensional gel analysis. After either silver staining (Maus et ai, 1995) or immunoblot analysis (Smith et al., 1987 Havel et at., 1992), interphase lamins Dmi and Dm2 can be detected as multiple spots, indicating multiple posttranslationally modified forms. Meiotic lamin Dmmit in the stage 14 oocyte extract exhibits an entirely different pattern (Maus et ai, 1995). When embryo nuclei, for example, are added to an oocyte disassembly extract and incubated for 90 min, a pattern distinct from the interphase lamins and indistinguishable from that of the extract alone should be seen, indicating that embryo nuclear lamin was converted to forms highly similar to, if not identical to, meiotic lamin Dm ,ii. 

Figure 4 Human platelet proteome. Proteins from human platelets (100 pg protein) were separated by two-dimensional gel electrophoresis using isoelectric focusing in the first dimension in a pH 3-10 gradient and SDS-PAGE in the second using a 10% 1.5 mm gel followed by silver staining. The relative molecular masses (H) and isoelectric points of the proteins (p/) separated are indicated. (Courtesy of Maguire PB, Royal College of Surgeons of Ireland.)...

Two-dimensional electrophoresis [86] is a well established technique for the separation of intact proteins. In the first dimension the proteins are separated based on their isolectric point while the second dimension separates them based on their size. The presence on the gel of the proteins is revealed by Coomassie blue or silver staining. Under favorable conditions several thousand spots can be differentiated. The gel is digitized and computer-assisted analysis of the protein spot is performed. The spots of interest are excised either manually or automatically and then digested with trypsin. Trypsin cleaves proteins at the C-terminal side of lysine and arginine. In general one spot represents one protein and the peptides are analyzed by MALDI-TOF to obtain a peptide mass fingerprint. A peptide mass fingerprint involves the determination of the masses of all pep-... 

Fig. 3. Two-dimensional analysis of PDZ proteins interacting with the 5-HT2A and the 5-HT2C receptors C-termini. (A) Proteins from mice brain that bind to the C-terminus of the last 14 residues of the receptors were separated on 2D gels and stained with silver. Proteins that interact specifically (directly or indirectly) with the PDZ ligand of the receptor (arrows) were detected comparing protein patterns obtained with the native peptides (see Fig. 1) and mutant peptides in which the last residue was replaced by alanine. The position of one protein retained in a PDZ-independent manner by the 5-HT2A receptor C-terminus is also indicated (arrowhead). (B) Molecular determinants in the C-terminus of 5-HT2A receptor involved in its preferential interaction with CIPP.

Figure 9.12. Comparison of identical polyacrylamide gels stained with Coomassie Blue (lanes 1-3) and Silver Stain (lanes 4-6). The initial sample concentrations are identical for lanes 1 and 4, 2 and 5, and 3 and 6. [Reprinted, with permission, from B. S. Dunbar, Two-Dimensional Electrophoresis and Immunological Techniques , Plenum Press, New York, 1987. 1987 Plenum Press.]...

Figure 1. A two-dimensional electrophoresis (2DE) separation of 80 tg of heart (ventricle) proteins. The first dimension comprised an 18 cm non-linear pH 3-10 immobihsed pH gradient (IPG) subjected to isoelectric focusing. The second dimension was a 21 cm 12% SDS-PAGE (sodium dodecylsulphate polyacrylamide gel electrophoresis) gel. Proteins were detected by silver staining. The non-linear pH range of the first-dimension IPG strip is indicated along the top of the gel, acidic pH to the left. The Mr (relative molecular mass) scale can be used to estimate the molecular weights of the separated proteins.

Figure I. Details of silver stained high-resolution two-dimensional polyacrylamide gel of (A) plasma, and (B) of serum. Note the absence of the spots corresponding to prothombin in panel B. First dimension immobilized 4 to 7 pH gradient, second dimension 9 to 16% polyacrylamide gel electrophoresis.

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