Protein patterns, visualization electrophoresis

The protein components of a membrane can be readily visualized by SDS-polyacrylamide gel electrophoresis. As discussed earlier (Section 4.1.4). the electrophoretic mobility of many proteins in SDS-containing gels depends on the mass rather than on the net charge of the protein. The gel-electrophoresis patterns of three membranes—the plasma membrane of erythrocytes, the photoreceptor membrane of retinal rod cells, and the sarcoplasmic reticulum membrane of muscle—are shown in Figure 12.16. It is evident that each of these three membranes contains many proteins but has a distinct protein composition. In general, membranes performing different functions contain different repertoires of proteins. 

Analyzing complex protein patterns by 2D gel electrophoresis has been a research tool since the early 1970s51,68. With this method it is possible to separate and visualize over 1000 distinct proteins in one experiment. Proteins are separated in the first dimension by isoelectric focusing (in a gel that separates proteins based on their relative amounts of acidic and basic amino acids) and in the second dimension by size. The proteins are visualized by staining and then quantified by densitometry. Figure 6 contains an example of a silver-stained 2D-PAGE analysis of liver proteins obtained from control and E2-treated largemouth bass. By visual inspection it is clear that there are numerous proteins expressed in the treated sample that are absent in the control, and there are proteins in the control that are not present in the treated sample. These spots would all be candidates for protein identification. 

The total proteins synthesized by bacteria grown under standard conditions, when analyzed by polyacrylamide gel electrophoresis (PAGE), form patterns that can be compared to those of known strains by visual or computer-assisted... 

To determine the values for Ton, T aik, and Toff for these four species, isolated sections of gill tissue were exposed to a series of temperatures and then incubated in 35S-methionine/ 35S-cysteine to allow monitoring of patterns of protein synthesis. Newly synthesized proteins were separated by gel electrophoresis and visualized using autoradiography (figure 7.11). As illustrated for gills of 13°C-acclimated T. funebralis, strong induction of several hsp s... 

Coomassie brilliant blue (CBB) stain is more sensitive than Amido Black or Ponceau S and is widely used. The concentrations of many proteins are too low to be seen as distinct stained bands, or they are overshadowed by proteins of higher concentrations that migrate near them. In addition, some proteins stain poorly because they contain high proportions of lipid (lipoproteins) or carbohydrate (AAG). Densitometry may be used for rough quantification of individual bands and for graphic displays of stained electrophoresis patterns, but visual examination by a trained observer is much preferred., I. / ... 

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