Detection silver-based stains

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. 

Ultrasensitive Silver-Based Stains for Protein Detection... 

Detection methods based on the post-electrophoretic staining of proteins with fluorescent compounds have the potential of increased sensitivity combined with an extended dynamic range for improved quantitation. The most commonly used reagents are the SYPRO series of dyes from Molecular Probes (Patton, 2000). Extensive studies have been carried out to evaluate the sensitivity of these fluorescent dyes compared with silver staining (Berggren et al., 2002 Lopez et al., 2000). In our laboratory (Yan et al.,... 

Glyoxal-based fixatives work faster than formalin. Small biopsies may be ready to process after only an hour while properly grossed larger specimens are ready in about 6h. Structural detail is remarkable in its clarity (Fig. 12.9). Red blood cells are lysed, but that rarely presents a problem. Eosinophilic granules are reduced in prominence (see below). Special stains work well, except for tests for iron (the mildly acidic pH is detrimental) and the silver detection methods for Helicobacter pylori. Most notably, glyoxal-fixed tissues retain strong immunoreactivity for most antigens. The chemistry behind most of this is known. 

Duan, L., Wang, Y., Li, S.S-C., Wan, Z., and Zhai, J. (2005) Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method. BMC Infect. Dis. 5, 53. http //www.biomedcentral. com/1471-2334/5/53. 

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). 

The search for more rapid and sensitive methods of protein detection after electrophoresis led to the development of fluorescent staining techniques. Two commonly used fluorescent reagents are fluorescamine and anilinonaphthalene sulfonate. New dyes based on silver salts (silver diamine or silver-tungstosilicic acid complex) have been developed for protein staining. They are 10 to 100 times more sensitive than Coomassie Blue (Fig. 4.7). 

This approach was pioneered by the group of Yates [43]. Using standard mixtures of proteins, they established the proteins in the mixtures with 30-fold difference in molar quantity can still be successfully identified. Initially, the method was applied to protein mixtures obtained by immunoaffinity precipitation or similar specific isolation procedures based on protein interaction (Ch. 17.4.1). In another study, E. coli periplasmic proteins were identified by the same approach [44]. The protein fraction was enriched using anion-exchange chromatography (AEC). Part of each fraction was separated by GE. The proteins were detected by silver staining. 

Silver staining of proteins after their separation by electrophoresis is based on binding of silver ions to sulfhydryl and carboxyl groups of proteins. The proteins are detected as black precipitate of silver. The sensitivity of this method is 20-100 times more sensitive than Coomassie blue staining. 

Figure 12.11 Chip-based electrical detection of target DNA strands using DNA-AuNP probes. Immobibzed capture strands on the chip are hybridized with the target strands and DNA-AuNP probes followed by silver staining enhancement, which changes the electric conductance for target detection. (Reproduced with permission from S.-J. Park et al., Science 2002, 295, 1503-1506. Copyright 2002 American Association for the Advancement of Science.)...

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