Amino acids silver staining

Amino groups involved in peptide bonding and N-terminal amino groups are in themselves insufficient for visualization with silver stain. If they were capable of independently reducing silver ions, all peptides, proteins, and amino acids would stain positively. However, the amino groups involved in peptide bonding and N-terminal atoms may be of some importance for the intensity of the stain, as these atoms have been observed to form 13 different complexes with copper between pH 1.5 to pH 11.0 (47,). Bound copper may be reduced under the conditions some stain protocols and then be displaced by silver. Alternatively, silver may also interact directly, but weakly, with these groups. 

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). 

Analyzing complex protein patterns by 2D gel electrophoresis has been a research tool since the early 1970s51,68. With this method it is possible to separate and visualize over 1000 distinct proteins in one experiment. Proteins are separated in the first dimension by isoelectric focusing (in a gel that separates proteins based on their relative amounts of acidic and basic amino acids) and in the second dimension by size. The proteins are visualized by staining and then quantified by densitometry. Figure 6 contains an example of a silver-stained 2D-PAGE analysis of liver proteins obtained from control and E2-treated largemouth bass. By visual inspection it is clear that there are numerous proteins expressed in the treated sample that are absent in the control, and there are proteins in the control that are not present in the treated sample. These spots would all be candidates for protein identification. 

The amount of proteins present in a large number band separated on 2D gel can be measured, and their relative abundance can be established by a variety of methods. First, the different samples of proteins are separated on 2D gel, and then the intensity of protein bands is measured by the intensity of dyes used to visualize the bands. The intensity of protein bands can be measured by a densitometric scan. Staining with silver stain is sensitive. Staining with certain fluorescent dye is equally useful. Alternatively, proteins in two cell types are labeled by growing cells in the presence of radioactive amino acids, such as methionine containing S35 sulfur. The protein samples obtained from the two cell types are then separated by 2D gel. The protein bands are visualized as spots on an X-ray film placed on the gel. The intensity of each spot on the film is determined by densitometry. 

Previous studies have reported silver staining with other amino acids. Heukeshoven and Dernick reported silver staining of the homopolymers of glycine, serine, proline and aspartic acid (4 ) while Nielsen and Brown reported the formation of colored silver complexes with aspartate, and tyrosine (45,). Staining of these homopolymers was not observed in the study of Merril and Pratt (32,), and prior metal binding studies failed to demonstrate metal interactions with the side-chain hydroxyl groups of serine, threonine or tyrosine... 

These discrepancies concerning the non-basic amino acids may be due to differences in the staining procedures employed the Heukeshoven and Dernick study stained homopolymers on polyacrylamide gel, Nielsen and Brown studied formation of silver-amino acid complexes in solution. Both of these studies used formaldehydye in an alkaline sodium carbonate solution for image development, while Merril and Pratt utilized acidic conditions and a combination of light, hydroquinone and formaldehyde for image formation (32). 

The importance of the basic amino acids has been further substantiated by evaluations of the relationship between a denatured protein s amino acid mole percentages and its ability to stain with silver. The best correlations were achieved when a comparison was made between the slope of the linear portion of a denatured protein s staining curve and the protein s mole percentages of the basic amino acids, histidine and lysine ( ). A similar correlation was observed by Dion and Pomenti(4 ). Dion and Pomenti suggested that this correlation may be due to an interaction between lysine and glutaraldehyde, which was used in their stain protocol. The bound glutaraldehyde could supply aldehyde groups to facilitate the reduction of ionic silver. While this mechanism may play a role in... 

The chemistry of silver staining is not entirely known but a recent review, (50 ). reaffirms that basic and sulfur-containing amino acids contribute in a substantial way to the staining reaction. The exact nature of the chemistry of formation of nucleation centers for silver reduction in proteins is still being investigated. 

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