Staining techniques, electrophoresis

Castelianos-Seeea, L, Hardy, E., Guerra, M., Estevez, E., Mehl, E., Eeank, R. W. (1998). Understanding the mechanism of the zinc-ion stains of biomacromolecules in electrophoresis gels generalization of the reverse-staining technique. Electrophoresis 19, 2398-2406. 

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. 

Figure 2.1. Schematic illustration oftwo-dimensional gel electrophoresis. Proteins are extracted from the organism of interest and solubilized. The first dimension separates proteins based on isoelectric point. The pi strip is reduced and alkylated and applied to an SDS-PAGE gel for separation by molecular weight. Proteins canbe visualized using a number of staining techniques.

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). 

Recently Heremans demonstrated mucopolysaccharides in urine after zone electrophoresis by means of an Alcian blue-acid fuchsin staining technique (HI).3 Applied in our laboratory to a curtain after a run of serum, two fractions, references -)- 86.30 and - - 86.12, were demonstrated but do not seem to be regular components of serum, at least not in that high concentration (Fig. 62c). 

Fig. 62a, b, and c. Some staining techniques adapted to paper electrophoresis. Sample human serum. Run continuous electrophoresis. The fractions are labeled according to the proposed reference system (see text). The known fractions are +86.20 Albumin +86.18 oij-globulin +86.12 a2-globulin +86.07 3-globulin —86.02 Y-globulin. 

Apart from the wide ran of staining techniques, fluore nt and radioactive labels are often used. FITC is used for protein detection following SDS-electrophoresis, e.g. Rat hemoglobin fractions have been assayed by labeling with Double labeling with and makes it possible to detect proteins from two different... 

Hb is another analyte, which is easily amenable to CE analysis. This is because of its high clinical concentrations as well as its homogeneity. UV detection (absorption of 415 nm visible light) of Hb is more accurate than the dye-staining technique for protein quantitation. Hb variants such as HbS, HbC, and HbA (normal control) were subjected to CE analysis. With an optimized running buffer, the three species of Hbs were separated as three distinct peaks in the electropherogram (Fig. 3a) these results are comparable to conventional gel electrophoresis (Fig. 3b). >... 

Lipopolysaccharides prepared from strains of Serratia marcescens that are resistant to polymyxin have higher carbohydrate and lower protein contents than do those of normal strains, both before, and after, treatment with the antibiotic.The converse is true of lipopolysaccharides obtained from cells sensitive to polymyxin B. Two lipopolysaccharides (one of mol. wt. 1.2 x 10 ) were detected after polyacrylamide gel electrophoresis of the outer membranes of S. marcescens Staining with a carbocyanine dye was found to be more sensitive and specific for lipopolysaccharides than other staining techniques in general use. 

Zlotnick, G. W. Gottlieb, M. A sensitive staining technique for the detection of phosphohydrolase activities after polyacrylamide gel electrophoresis. Anal. Biochem. 1986, 153, 121-125. 

We have isolated reaction centers from Rhodospirillum rubrum G- 9 cells labeled with the isotope 39-Fe. On LDS-polyacrylamide electrophoresis the preparation shows the typical three bands H, M and L and some minor contaminations (Figure 1). The specific staining technique for c-type cytochromes gave no positive reaction, indicating that there is no such cytochrome among the contaminations (datanot shown). 

Most sample components analyzed with electrophoretic techniques are invisible to the naked eye. Thus methods have been developed to visualize and quantify separated compounds. These techniques most commonly involve chemically fixing and then staining the compounds in the gel. Other detection techniques can sometimes yield more information, such as detection using antibodies to specific compounds, which gives positive identification of a sample component either by immunoelectrophoretic or blotting techniques, or enhanced detection by combining two different electrophoresis methods in two-dimensional electrophoretic techniques. 

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