Specificity, silver stains

Figure 14 6 Silver-stained SDS-PAGE gel of PatA binding proteins. Lane 1, sample 1 nonspecific proteins captured by the streptavidin-agarose resin Lane 2, sample 2 proteins affinity captured by the presence of B-Pat A Lane 3, sample 3 affinity capture of target proteins was blocked by prior addition of free PatA before incubation with B-PatA. The two arrows point to two proteins specifically detected in sample 2 versus sample 1, which were also lost due to competition in sample 3, with apparent molecular weights of 38 and 48 kDa.

This procedure is not solely specific for carbohydrate side chains of proteins. Unglycosylated proteins may also be stained. To identify glycosylated proteins, the sample should be run in at least two identical lanes cut the gel and stain a lane with the common protein silver stain (Protocols 2.4.2.1 to 2.4.2.4) and the other lane by the described method. Compare pattern and intensity to identify glycoproteins. Glycosylated macromolecules are also stainable with Schiff s reagent (Protocol 2.4.4.1), but with less sensitivity. 

PAS staining (periodic acid - Schiff s reagent) colors compounds with vicinal hydroxyl groups, i.e., mainly oligosaccharide side chains in glycoproteins, glycolipids, and nucleic acids. The sensitivity and stability is much lower than the silver staining Protocol 2.4.2.S but more specific. 

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). 

While it may appear to be a matter of semantics, the author has become convinced that whether IHC is viewed as a stain or as an assay , can play an important role in establishing the proper mind set of the laboratory staff and the pathologists. The IHC method is regarded by many as simply a stain it produces a visible tinctorial reaction within the tissue section. However, IHC should not be regarded as simply another special stain , like a PAS stain or a silver stain. As already noted, IHC is essentially an ELISA method applied to a tissue section. In this respect, when correctly performed, IHC has the potential to perform as a reproducible and quantitative tissue based ELISA assay much more than a simple stain. That the IHC method mostly does not perform to this level, reflects faults in the application of the method, specifically inconsistent sample preparation, lack of reference or calibration standards, and inadequate validation of reagents (7, 8). The use of RTUs does not finally solve these problems, but for reasons that will be discussed below, can lead to increased reproducibility and consistency in a practical... 

Fibroblasts were selected because they are readily cultivated and radio-labeled and are available from skin explants of a variety of species. Autoradiograms of radiolabeled cellular proteins are more diverse and easily analyzable for molecular systematics than alternatives such as silver-stained serum or erythrocyte protein patterns. We have achieved nearly 100% success rates at minimal discomfort and risk to human subjects by establishing cultures from 3 mm punch biopsies from the upper buttock. Local lidocaine anesthesia is used. Samples are collected following informed consent and under an approved human research protocol. Other species are sampled by dart gun8 or while sedated. Skin samples can be collected from various body sites without compromising the 2D electrophoresis metric, which does not rely on quantitative differences in protein expression. To comply with the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), tissues collected from wild and captive exotic animals must be obtained under specific permits issued by the U.S. Fish and Wildlife Service. 

Figure 10.3 Detailed view of the protein fraction specifically eluted by A 77 1726 from the affinity column. Analytical 10-17% gradient SDS-PAGE, silver-stained.

Because Coomassie staining is a slow process and it may interfere in MS analysis, silver staining has been proposed as an alternative [13]. Silver staining improves the detection limit from 50 ng to 1-10 ng It is easier to remove as it does not act by specific binding to the protein. 

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. 

The usual carcinoid tumor is sofid and yeUow-tan in appearance. Tumor cells exhibit a monotonous morphology, with pink granular cytoplasm and round nuclei with infrequent mitoses. Most carcinoids can be recognized by their reactions to silver stains and to neuroendocrine cell markers, such as chromogranin and neuron-specific enolase. Ultra-structuraUy, carcinoids possess numerous membrane-bound, electron-dense neurosecretory granules. These granules contain peptide hormones and bioactive amines, which can occasionally be identified by immunocytochemi-cal techniques. 

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