Silver Staining

Silver has been known to be able to develop images for over two hundred years, first being usefully exploited in photography and then rapidly adopted for use in histological staining procedures. The ability of silver to detect proteins following their separation by gel electrophoresis was first recognized by Merril and his colleagues in 1979. Subsequently, more than a hundred silver-staining procedures have been described and this group of methods has become the standard approach for the sensitive detection of gelseparated proteins. However, certain classes of proteins, such as calcium-binding proteins and glycopro-teins, stain rather poorly, with an inverse relationship between the intensity of silver staining and the proportion of the molecule that is composed of carbohydrate. Pre-staining with cationic dyes prior to silver staining can significantly improve the sensitivity of detection of glycoproteins.

Depending on the method, silver staining is between ten and a hundred times more sensitive than staining with CBB R-250, and is able to detect low nanogram amounts of protein. There can be problems in using silver staining as a quantitative procedure as it is known to be non-stoichiometric. However, staining intensity is linear over a 40-fold range, comparing well with the 20-fold linear range of CBB R-250. Above this limit, the stain becomes non-linear, resulting in saturation and even negative staining of bands and spots at very high protein concentrations, making quantitation of such protein zones impossible. In a two-dimensional electrophoresis study of human leukocyte proteins, over 200 spots were observed to have coefficients of variation less than or equal to 15% when data from replicate patterns were analysed. In dilution experiments, the majority ( > 80%) of the proteins were found to have a linear relationship between the amount of protein loaded and the spot volume. An additional problem with the quantitation of silver staining is that the relationship between staining intensity and protein concentration may be different for each protein. However, it is often forgotten that this is also the case for staining with CBB R-250.

All silver-staining procedures depend on the reduction of ionic silver to its metallic form, but the precise mechanism involved in the staining of proteins has not been fully established. It has been proposed that silver cations complex with protein amino groups, particularly the s-amino group of lysine, and with sulfur residues of cysteine and methionine. However, staining cannot be attributed exclusively to specific amino groups suggesting that some other component of protein structure is also responsible for differential protein staining.

Procedures for silver staining can be grouped into two main types depending on the chemical state of the silver when used for impregnating the gel. The first group comprises alkaline methods based on the use of ammoniacal silver or diamine solution, prepared by adding silver nitrate to sodium-ammonium hydroxide mixture. Copper can be included in these diamine methods to give increased sensitivity, probably by a mechanism similar to the Biuret reaction. The silver ions complexed to proteins in the gel are then developed by reduction to metallic silver with formaldehyde in an acidified environment, usually using citric acid. In the alternative group of methods, silver nitrate in a weakly acidic (around pH 6.0)

solution is used for gel impregnation. Development is subsequently achieved by the selective reduction of ionic silver to metallic silver by formaldehyde made alkaline with either sodium carbonate or sodium hydroxide. Any free silver is washed out of the gel prior to development to prevent precipitation of silver oxide that would result in high background staining.

The majority of silver staining procedures are monochromatic, resulting in dark brown to black protein zones. However, if the development time is extended with saturation of the zones of highest protein concentration, then colour effects can be produced. In a comparative study of several methods based on both the silver diamine and silver nitrate approaches, the most rapid procedures were found to be generally less sensitive than those which were more time-consuming. The use of glutaraldehyde pre-treat-ment of the gel and silver diamine as the silvering agent were found to be the most sensitive and example of a gel stained with a method of this type is shown in Figure 2.

Increasingly, proteins are being visualized in gels for subsequent identification and characterization by techniques such as mass spectrometry. In this case, glutaraldehyde cannot be used and silver-staining protocols that omit this reagent must be used. However, this modification results in a decrease in sensitivity and uniformity of staining as well as an increase in background.

It is a common experience that silver-staining procedures can give rise to problems when based on the m b c d e f 9

Figure 2 SDS-PAGE separation of human heart proteins (lanes b-g). Lane (a) contains the molecular weight marker proteins and the scale at the left indicates protein size in kDa. The gel has been silver stained. The sample protein loadings were (b) 1 |ig, (c) 5 |ig, (d) 10 |ig, (e) 25 |ig, (f) 50 |ig, (g) 100 |ig.

Figure 2 SDS-PAGE separation of human heart proteins (lanes b-g). Lane (a) contains the molecular weight marker proteins and the scale at the left indicates protein size in kDa. The gel has been silver stained. The sample protein loadings were (b) 1 |ig, (c) 5 |ig, (d) 10 |ig, (e) 25 |ig, (f) 50 |ig, (g) 100 |ig.

use of laboratory-prepared reagents. If care is not taken with the use of high-purity water, reagents and glassware, then problems of high background staining, surface 'mirror' effects and poor reproducibility can be experienced. Many of these problems can be alleviated using one of the commercially available silver-staining kits (for example from Amersham-Pharmacia Biotech, Bio-Rad Laboratories, Richmond, CA, USA).

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