Densitometry, silver-stained

Figure 5. A plot of the amount of nuclease complex formed in the continuous variation titration experiments versus mole fraction of the R subunit as determined by densitometry of silver stained gels such as that shown in figure 4. The lines drawn are the theoretical curves expected for the association of 1 (. ..), 2 (-), or 3 (—) R subunits per molecule of MjS, mtase. The error bars are +/- one standard deviation.

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. 

The use of silver-based stains increases the detection sensitivity up to 100 fold, with individual bands containing as little as lng of protein usually staining well. However, because silver binds to protein non-stoichiometrically, quantitative studies using densitometry cannot be undertaken. 

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