Proteomics silver staining

The second step in 2D electrophoresis is to separate proteins based on molecular weight using SDS-PAGE. Individual proteins are then visualized by Coomassie or silver staining techniques or by autoradiography. Because 2D gel electrophoresis separate proteins based on independent physical characteristics, it is a powerful means to resolve complex mixtures proteins (Fig. 2.1). Modem large-gel formats are reproducible and are the most common method for protein separation in proteomic studies. 

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). 

Sinha, P. Poland, J. Schnolzer, M. Rabilloud, T. A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis. Proteomics (Germany) 2001, 1(7), 835-840. 

Differential 2DE displays of post-gel labelled silver stained and pre-labelled S-35 proteins also represent a sensitive procedure to visualize and identify synthesized elements of proteomes, respectively. 

Widespread use of mass spectrometry to analyze large numbers of spots from two-dimensional gels has also brought out another apparent shortcoming of 2DE. Articles on proteome-wide analyses of Saccharomyces cerevisiae point to a growing awareness in the literature and community of another shortcoming of 2DE, namely that no spots with low abundance can be identified from silver stained two-dimensional gels. °-"... 

Figure 2. A 2D proteome map of L. major Friedlin. Whole cells were extracted with a lysis buffer containiug urea, thiourea, and CHAPS. Samples were run using a variety of narrow-range IPG strips (4.0-5.0, 4.5-5.5, 5.0-6.0, 5.5-6.7, and 6-11 Amersham), gels were silver-stained, and the images were merged using PDQuest software (BioRad).

Figure 4 Human platelet proteome. Proteins from human platelets (100 pg protein) were separated by two-dimensional gel electrophoresis using isoelectric focusing in the first dimension in a pH 3-10 gradient and SDS-PAGE in the second using a 10% 1.5 mm gel followed by silver staining. The relative molecular masses (H) and isoelectric points of the proteins (p/) separated are indicated. (Courtesy of Maguire PB, Royal College of Surgeons of Ireland.)...

White IR, Pickford R, Wood J, Skehel JM, Gangadharan B, Cutler P. A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis. Electrophoresis 2004 25(17) 3048-3054. 

Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G. M., Carnemolla, B., Orecchia, P., Zardi, L., and Rigetti, P. G. (2004) Blue silver a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25, 1327-1333. 

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