Methionine, silver-staining

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. 

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. 

Figure. 5. Formylation of HIV-1 p24 . (A) As shown in the upper spectrum, the major peaks of [M + H] at m/z 2016.17 represent the N-terminal tryptic peptide (Proi-Argig) of HIV-Ilav-1 p24 derived from silver staining gel. The other peaks of [M + H] at m/z 2032.13 and 2044.19 could be assigned to the methionine-oxidized form and the formylated form of the N-terminal tryptic peptide (Proi-Argig), respectively. As shown in the lower spectrum, these peaks were also detected in the spot corresponding to HIV-1lav-i p24 derived from Coomassie brilliant blue R-250 staining gel. The molecular mass of 28.02 corresponding to the formyl group was deleted. (B) The formyl group was sequentially cleaved with 0.6 N hydrochloric acid as described in Section 2.4.1.

Silver-stained polyacrylamide gels " " have been used in quantitative and fast separations of individual proteins labelled with weak -emitters H) by polyacrylamide gel electrophoresis. The amount of radioactivity in a given band can be determined rapidly by liquid scintillation counting, which was found to be independent of the silver deposition and total protein content, but hnear with respect to the amount of radioactivity in the given band, thus avoiding the lengthy exposures needed, for instance, in the autoradiography of [ H]methionine-labelled proteins separated by polyacrylamide gel electrophoresis. The method was used to determine [ H]mannose and [ H]fucose incorporated into a cell adhesion protein . 

Figure 3 A shows a silver-stained gel of electroeluted glutathione S-transferase ti (GST-tt) cut from a dry Coomassie brilliant blue-stained 2D gel of SV40-transformed keratinocytes. The sample was co-run with a small amount of [ S]methionine-...

S]-L-methionine labeling) and the protein accumulation (staining with silver nitrate) were overlaid in dual channel images. Proteins whose synthesis was induced in response to the stressor but have not accumulated yet are colored red, repressed spots that are still present but not synthesized anymore are colored green, and spots without changed expression behavior in response to stress are shown in yellow (Chapter 3, Fig. 5 see discussion on p. 40). 

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