Quantitation silver-stained

To quantitate proteins from staining, a densitometer aided by computer software is used to evaluate band areas of samples compared to band areas of a standard curve. Amido black, Coomassie Brilliant Blue, and silver stains are all appHcable for use in quantification of proteins. 

Jensen, K.F., Olin, J., Haykal-Coates, N., O Callaghan, J., Miller, D.B., and de Olmos, J.S., Mapping toxicant-induced nervous system damage with a cupric silver stain a quantitative analysis of neural degeneration induced by 3,4-methylenedioxymethamphetamine, NIDA Res. Monogr. 136, 133-149 discussion 150-154, 1993. 

If the purpose of gel electrophoresis is to identify low-abundance proteins (e.g., low-copy-number proteins in a cell extract or contaminants in a purification scheme), then a high protein load (0.1 to 1 mg/ml) and a high-sensitivity stain such as silver or fluorescence should be used. When the intention is to obtain enough protein for use as an antigen or for sequence analysis, then a high protein load should be applied to the gel and the proteins visualized with a staining procedure that does not fix the proteins in the gel, e.g., colloidal CBB G-250 (Subsection 8.2.8.1). Furthermore, for purposes of quantitative comparisons, stains with broad linear ranges of detection response should be used. 

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. 

Automatic and quantitative microscopes tend to give erroneous results for transparent particles. To overcome this problem Amor and Block [49] a silver staining technique to make the particles opaque. The particles are dry-mounted on to a thin film of tacky colloidon on a microscope slide. Silver is then deposited from solution using the silver mirror reaction. Preliminary sensitizing the crystalline surface ensures that much more silver is deposited on the particles than on the colloidon. A method of staining particles in aqueous solution prior to deposition on a membrane filter for analysis is also given. 

At the completion of 2-D PAGE, gels are fixed and the separated proteins visualized by any appropriate technique (e.g. silver staining, autoradiography). It then remains to extract qualitative and quantitative data from the resulting complex 2-D protein patterns. Simple visual inspection can provide only limited information and it is usually necessary to use sophisticated computer analysis systems (71, 106, 10 7). 

Quantitative Inter-oel Protein Comparisons The occurance ot Protein specitic staining curves with silver staining requires that quantitative inter-gel comparative studies limit comparisons to homologous protein bands or spots on each gel. For example, the actin spot on one gel can be compared with an actin spot on another gel, but not with a transterrin spot. These limitations to homologous comparisons are also applicable to most of the organic stains, including the Coomassie Blue stains, (72). 

Color staining of the gel with GELCODE reveals protein patterns which are easily interpreted for qualitative analysis. The color aids in the analysis by distinguishing overlapping spots or bands which inherently have similar or identical isoelectric points or molecular weights. Quantitative analysis of color silver stained gel... 

It has been repeatedly reported that silver stained methods are not suitable for computerized quantitation because of their capriciousness and nonlinearity. This is apparently true of the stains based on reduction with citric acid and weak carbonate because there is no predicting the slope of a plot of integrated intensity versus protein concentration without the use of a reliable and reproducible color. Thus, in order to use quantitation with these two methods one must perform a standard curve with each protein as its own standard or accept some relative standard for normalization. The relative approach has been successfully used with GELCODE and allowed measurement of protein changes within an experimental protocol (1 ). These disadvantages have discouraged the acceptance of silver staining to its full potential application. 

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.,... 

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 . 

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