Sensitivity, silver stains

For visualisation of proteins after separation on gel, one could use different stains such as Coomassie blue stain or more sensitive silver staining. The Coomassie blue staining is relatively less sensitive than silver staining, but is highly convenient to use. 

Figure 10.7 Polypeptide patterns of material associated with lipid globules of cow s milk. The sodium dodecyl sulfate-polyacrylamide gel in (a) was stained with coomassie blue, and polypeptides in the gel in (b) were visualized with the more sensitive silver stain (Merril ef at. 1981). In both (a) and (b) the left lane contains proteins extracted directly from washed milk...

Merril, C. R., Dunau, M. L. and Goldman, D. 1981. A rapid sensitive silver stain for polypeptides in polyacrylamide gels. Anal Biochem. 110, 201-207. 

Tsai, C.M., Frasch, C.E. A sensitive silver stain for detecting Hpopolysaccharides in polyacrylamide gels. Anal Biochem 119 (1982) 115-119. 

The SDS-PAGE method of Laemmli (1970) is convenient to verify the purity of the Ig, particularly with the very sensitive silver staining method (Section 16.1.1). It is good practice to analyse both unreduced and reduced (with 2-ME) samples. 

Using the more sensitive silver stain, however, Goldman and coworkers (G9) have been able to resolve more than 300 proteins and/or peptides (denatured sample) in csf using lEF/SDS-PAGE (Fig. 14). Twenty six of the proteins have been tentatively identified (Fig. 15), and comparison of the protein patterns with the corresponding plasma protein patterns has revealed several clusters of unidentified proteins that were more prominent in csf than in plasma and hence, may be of CNS origin. 

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

Sensitivity Silver stains currently offer the most sensitive non-radioactive method for detecting proteins separated by gel electrophoresis. They are 100-fold more sensitive than the Coomassie stains for most proteins (15-16). Chemical-development silver stains are in general, more sensitive than photo-development silver stains. This loss in senstivity may be compensated for by the ability of photo-development stain to produce an image within 10 to 15 minutes... 

In our laboratory experience, we have found that many commercially available proteins are not as pure as they are purported to be. Using sensitive silver staining processes and the resolving power of the 2-dimensional technique to examine purchased standards, we have observed multiple forms of purchased proteins. It appears that many companies simply blend proteins to provide major bands for molecular weight controls. Unfortunately, breakdown products and protein impurities may be easily observed. Care must be taken in using these proteins as standards for comparison purposes. 

The sample bands are not usually visible during electrophoresis (unless intrinsically coloured or fluorescent), but are normally visualized afterwards by immersing the gel in specific staining solutions. For DNA samples, intercalating fluorescent dyes such as ethidium bromide (see Section 2.4) are used, whereas proteins can be visualized using histological stains such as Coomassie blue or more sensitive silver stains. 

Tegelstrom H (1986) Mitochondrial DNA in natural populations. An improved routine for the screening of genetic variation based on sensitive silver staining. Electrophoresis 7 226-230... 

Merril, C. R., Switzer, R. C., and Van Keuren, M. L. (1979) Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain. Proc. Natl. Acad. Sci. USA 76, 4335-4339. 

Polyacrylamide gel electrophoresis (PAGE) was performed as described (15,16). PAGE of reaction centers gave the following results Mildly treated reaction centers (incubation at 50 C) resulted in the LM-reaction center polypeptides resolved as one complex with M of about 56 kD. Denaturation at lOO C for 10 minutes resulted in separation of the L and M subunits with M values of 27 kD and 29 kD, respectively. Some minor contaminations were often detected by the very sensitive silver staining method. The most dominant contamination has a M of about 10 kD. This protein is not a cytochrome as can be concluded from the absorption spectrum, it rather is a non-coloured component. 

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