Silver stains, protein detection

Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1980). A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105, 361-363. 

Switzer, R. C., Ill, C. R. Merril, and S. Shifrin. 1979, A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal.Biochem. 98 231-237. 

More than a hundred times higher detection sensitivity, up to 0.1 ng of protein, is achieved by silver staining. It is based on reduction of silver ions to metallic silver due to differences in the reduction-oxidation potential between the sites occupied by proteins and the adjacent sites of the gel or membrane. Densitograms of silver-stained protein electro-phoregrams are linear over a 30- to 40-fold range for most proteins, but the slope of the response also differs for different proteins. 

Ohsawa, K, and Ebata, N., 1983, Silver stain for detecting 10 femtogram quantities of protein after polyaciylamide gel electrophoresis. Anal. Biochem., 135 409 15. 

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). 

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. 

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.

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. 

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... 

Place test and reference solutions, contained in covered test-tubes, in a waterbath for 2 min. Apply 10 ul of reference solution (f) and 50 pX of each of the other solutions to the stacking gel wells. Perform the electrophoresis under the conditions recommended by the manufacturer of the equipment. Detect proteins in the gel by silver staining. 

The minimal amount of applicable protein depends on the detection method. Mosdy detection by Coomassie staining is tenfold less sensitive than by silver staining, which drops down to 1 ng per band. 

A variety of methods are available to detect proteins separated by electrophoresis or to measure the concentration of total protein in a solution. These methods are normally based on the binding of a dye to one of the amino acids in protein, or a color reaction with an amino acid side chain. The most commonly used stains for protein detection on gels are Coomassie Brilliant Blue (98) and silver stain (99,100). These methods detect any protein residues, either in solution or on an electrophoresis gel. Their main requirement is sensitivity, not specificity. New, more sensitive dyes are being developed for the proteomic analysis of protein structure and sequence, for example Ruby Red (101). 

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