Staining silver

In silver staining, the gel is Impregnated with soluble silver ions and developed by treatment with formaldehyde, which reduces silver ions to form an insoluble brown precipitate of metallic silver. This reduction is promoted by protein. 

Jackson et al. (1988) were the first to employ the immunogold-silver staining (IGSS) method in combination with microwave heating. They completed within minutes the incubations in primary and secondary antibodies for detecting immunoglobulins in paraffin sections of human tonsil, van de Kant et al. (1990) applied the same method, except that resin instead of paraffin sections were used to detect bromodeoxyuridine incorporated in cells of the mouse testis. Tissue morphology is preserved better in a resin than in paraffin. The former also allows the use of thinner sections. 

Tissues are fixed with buffered formalin or Kryofix and embedded in paraffin (Boon et al., 1991). Sections (5 xm thick) are transferred to a glass slide, deparaffinized, rehydrated, and washed in running tap water for 10 min. They are treated with Lugol s iodine for 5 min and rinsed briefly in tap water. Following destaining with 2% aqueous sodium thiosulfate for 10-15 sec, the sections are washed in running tap water for 10 min. 

The sections are washed in two changes of 5 min each in Tris buffer I (2.5% NaCl and 0.55% Tween 20, diluted in 0.05 mol/liter Tris-HCl buffer, pH 8.2). The excess buffer is removed by wiping around the sections, which are then covered for 10 min with 75 pi of normal goat serum (NGS) diluted 1 1 with PBS (pH 7.4). Excess NGF is removed by wiping around the sections. 

Excess NGS is removed, and the sections are covered with 100 pi of colloidal gold conjugated secondary antibody. The slide is placed on the polystyrene platform in the microwave oven and heated at 50% for 5 min the oven contains a water load of 200 ml of tap water. The sections are washed in three changes of 5 min each in Tris buffer n, rinsed with distilled water, and washed three times of 3 min each in distilled water. 

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Amido black is a commonly used stain, but it is not very sensitive. It is often used to visualize concentrated proteins or components that are readily accessible to dyes such as proteins that have been transferred from a gel to nitrocellulose paper. Two of the more sensitive and more frequently used stains are Coomassie Brilliant Blue (R250 and G250) and silver stains. Because these stains interact differently with a variety of protein molecules, optimization of the fixative and staining solutions is necessary. The Coomassie stains are approximately five times more sensitive than amido black and are appropriate for both agarose and polyacrylamide gels. The silver stain is approximately 100 times more sensitive than Coomassie and is typically used for polyacrylamide gels. 

A silver stain is used when proteins exist in a very small quantity or when analysis of as many bands as possible created by separation techniques is desired. One positive apphcation of silver stain is its sensitivity. A drawback of the silver stain, however, is that it is more complex and often requires more troubleshooting to obtain the desired results. 

To quantitate proteins from staining, a densitometer aided by computer software is used to evaluate band areas of samples compared to band areas of a standard curve. Amido black, Coomassie Brilliant Blue, and silver stains are all appHcable for use in quantification of proteins. 

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.

The purity of the AE fraction was investigated by SDS-PAGE using Pharmacia PhastSystem with 10 - 15% SDS-gradient gels. Electrophoresis and silver staining of the proteins were performed as described by the manuals from Pharmacia. For determination of pi lEF 3-9 PhastSystem gels were used. 

Figure 5 shows the pattern of lyase isoenzymes along the purification process at first, three bands with lyase activity (pis 9.20, 9.00 and 8.65) were detected in the ammonium sulfate precipitate (B 1) in the peak eluted from the Superdex 75HR1030 column, only one band with lyase activity was detected, that correspond to the PNL with pi 9.20 (B 2), but more proteins were detected by silver staining (A 2). 

Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 nun) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 pg of protein in 10 pi of 1 % glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HCl buffer at pH 8.0 with CaClj M.- Broad pi Calibration Kit protein (Pharmacia), samples of 5 pg of protein were applied. 1.-Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.

Figure 1. a - SDS-PAGE of fraction T (silver staining), lane 1 fraction T, lane 2 ovalbumine and lane 3 molecular weight standards. 

IGSS Immuno-gold silver stain IHC Immunohistochemistry IHES Idiopathic hypereosinophilic syndrome... 

Marshall, T. and Williams, K. M. (1987). Electrophoresis of honey Characterization of trace proteins from a complex biological matrix by silver staining. Anal. Biochem. 167,301-303. 

M6. Maekawa, M., Sudo, K., Kitajima, M., Matsuura, Y Li, S. S.-L., and Kanno, T., Detection and characterization of new genetic mutations in individuals heterozygous for lactate dehydroge-nase-B (H) deficiency using DNA conformation polymorphism analysis and silver staining. Hum. Genet. 91, 163-168 (1993). 

The second step in 2D electrophoresis is to separate proteins based on molecular weight using SDS-PAGE. Individual proteins are then visualized by Coomassie or silver staining techniques or by autoradiography. Because 2D gel electrophoresis separate proteins based on independent physical characteristics, it is a powerful means to resolve complex mixtures proteins (Fig. 2.1). Modem large-gel formats are reproducible and are the most common method for protein separation in proteomic studies. 

Neukirchen et al. (1982) from the Max-Planck Institute employed a similar miniaturized IEF/SDS-PAGE system that was roughly 2 cm x 2 cm. Silver staining was used to detect spots containing as little as 10 pg of protein, and electrophoresis was used to separate the proteins contained within a single Drosophila egg. 

Jensen, K.F., Olin, J., Haykal-Coates, N., O Callaghan, J., Miller, D.B., and de Olmos, J.S., Mapping toxicant-induced nervous system damage with a cupric silver stain a quantitative analysis of neural degeneration induced by 3,4-methylenedioxymethamphetamine, NIDA Res. Monogr. 136, 133-149 discussion 150-154, 1993. 

Figure 14 6 Silver-stained SDS-PAGE gel of PatA binding proteins. Lane 1, sample 1 nonspecific proteins captured by the streptavidin-agarose resin Lane 2, sample 2 proteins affinity captured by the presence of B-Pat A Lane 3, sample 3 affinity capture of target proteins was blocked by prior addition of free PatA before incubation with B-PatA. The two arrows point to two proteins specifically detected in sample 2 versus sample 1, which were also lost due to competition in sample 3, with apparent molecular weights of 38 and 48 kDa.

Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850—858. 

De Waele, M., Renmans, W., Segers, E., De Valck, V., Jochmans, K., and van Camp, B. (1989) An immunogold-silver staining method for detection of cell surface antigens in cell smears. J. Histochem. Cytochem. 37, 1855-1862. 

Duan, L., Wang, Y., Li, S.S-C., Wan, Z., and Zhai, J. (2005) Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method. BMC Infect. Dis. 5, 53. http //www.biomedcentral. com/1471-2334/5/53. 

Ellis, I.O., Bell, J., and Bancroft, J.D. (1988) An investigation of optimal gold particle size for immuno-histological immunogold and immunogold-silver staining to be viewed by polarized incident light (EPI polarization) microscopy./. Histochem. Cytochem. 36, 121-124. 

Gillitzer, R., Berger, R., and Moll, H. (1990) A reliable method for simultaneous demonstration of two antigens using a novel combination of immunogold-silver staining and immunoenzymatic labeling. J. Histochem. Cytochem. 38, 307-313. 

Holgate, C., Jackson, P., Cowen, P., and Bird, C. (1983) Immunogold-silver staining new method of immunostaining with enhanced sensitivity./. Histochem. Cytochem. 31, 938. 

Moeremans, M., Daneels, G., Van Dijck, A., Langanger, G., and De Mey, J. (1984) Sensitive visualization of antigen-antibody reactions in dot and blot immuno overlay assays with the immunogold and immu-nogold/silver staining./. Immunol. Meth. 74, 353-360. 

An increase in sensitivity is realized by silver-staining, where residues containing sulfur (cysteine, methionine) or basic side chains (arginine, lysine, histidine) reduce Ag+, leading to brown or black colored bands. Here, down to 0.1 ng of protein can be detected. 

A modified version of 2DE and gel image analysis, with silver staining, autoradiography, and protein identification and measurement of peptide mass, uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as a rapid and sensitive technique for identifying peptides. MALDI-TOF-MS applies well to protein detection in biological fluids.56 A second advantage of this technique is... 

Cassab GI, Varner JE. Immunocytolocalization of extensin in developing soybean seed coats by immunogold-silver staining and by tissue printing on nitrocellulose paper. J Cell Biol 1987 105 2581-2588. 

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