Silver electrophoresis stain

Detection of proteins on thin-layer plates, gel slabs, or membranes is often accomplished by staining with a dye,265-267 the most widely used being Coomassie brilliant blue.268 Various silver-containing stains may also be used. After separation of a protein mixture by electrophoresis and transfer to an inert membrane,... 

Rochette-Egly, C., and Stussi-Garaud, C. (1984). Selective detection of calmodulin in polyacrylamide gels by double staining with Coomassie Blue and silver. Electrophoresis 5, 285-288. 

The purity of the AE fraction was investigated by SDS-PAGE using Pharmacia PhastSystem with 10 - 15% SDS-gradient gels. Electrophoresis and silver staining of the proteins were performed as described by the manuals from Pharmacia. For determination of pi lEF 3-9 PhastSystem gels were used. 

Marshall, T. and Williams, K. M. (1987). Electrophoresis of honey Characterization of trace proteins from a complex biological matrix by silver staining. Anal. Biochem. 167,301-303. 

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. 

Neukirchen et al. (1982) from the Max-Planck Institute employed a similar miniaturized IEF/SDS-PAGE system that was roughly 2 cm x 2 cm. Silver staining was used to detect spots containing as little as 10 pg of protein, and electrophoresis was used to separate the proteins contained within a single Drosophila egg. 

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. 

Figure 3 Biosynthesis and purification of 90-kD elastin analogue analyzed by denaturing polyacrylamide gel electrophoresis (10-15% gradient, visualized by silver staining). Lanes 1-7 time course of target protein expression at 0, 30, 60, 90, 120, 150, and 180 minutes after induction. Lane 9 soluble lysate of induced E. coli expression strain BLR(DE3)pRAMl. Lanes 10-13 protein fractions obtained from immobilized metal affinity chromatography of the lysate on nickel-NTA agarose (imidazole gradient elution). Lanes 8,14 protein molecular weight standards of 50, 75, 100, and 150 kD.

Two-dimensional electrophoresis [86] is a well established technique for the separation of intact proteins. In the first dimension the proteins are separated based on their isolectric point while the second dimension separates them based on their size. The presence on the gel of the proteins is revealed by Coomassie blue or silver staining. Under favorable conditions several thousand spots can be differentiated. The gel is digitized and computer-assisted analysis of the protein spot is performed. The spots of interest are excised either manually or automatically and then digested with trypsin. Trypsin cleaves proteins at the C-terminal side of lysine and arginine. In general one spot represents one protein and the peptides are analyzed by MALDI-TOF to obtain a peptide mass fingerprint. A peptide mass fingerprint involves the determination of the masses of all pep-... 

Tests of Purity, Isoelectric focusing (lEF) in a 0.5-mm thick horizontal slab gel was performed with LKB pH 7-9 ampholyte (Ampholine 1809-136) (5). Electrophoresis was run at 10°C for 6 h at a constant voltage of 1800 V. Protein was visualized using silver stain (9) or Sigma Coomassie Brilliant Blue G-250. 

Electrophoresis. Electrophoresis was conducted on 9 to 18% gradient gels under denaturing conditions as described (11,12). For detection, proteins were electrotransferred to nitrocellulose paper (13) and stained using colloidal gold with a silver overlay (14). 

Place test and reference solutions, contained in covered test-tubes, in a waterbath for 2 min. Apply 10 ul of reference solution (f) and 50 pX of each of the other solutions to the stacking gel wells. Perform the electrophoresis under the conditions recommended by the manufacturer of the equipment. Detect proteins in the gel by silver staining. 

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. 

Yan, J. X. Wait, R. Berkelman, T. Harry, R. A. Westbrook, J. A. Wheeler, . H. Dunn, M. J. A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 2000, 21(11), 3666-3672. 

A silver-stained gel obtained by electrophoresis of a standard protein mixture under denaturing conditions. Samples were run on 12% polyacrylamide gels,... 

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). 

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