Protein patterns, visualization

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. 

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). 

Comparison of relative protein expression levels between chromatographic runs can be performed using the clustering algorithm Cluster 3.0 and the patterns visualized in a heat map format using Java Treeview freeware software. 

After transfer is complete, remove the transfer sandwich. The gel can be stained in Coomassie blue to assess efficiency of transfer, and the nitrocellulose membrane may be stained with Ponceau S (Protocol 6) to visualize the total protein pattern prior to immunoblotting (Protocol 7). 

A regularly formed crystal of reasonable size (typically >500 pm in each dimension) is required for X-ray diffraction. Samples of pure protein are screened against a matrix of buffers, additives, or precipitants for conditions under which they form crystals. This can require many thousands of trials and has benefited from increased automation over the past five years. Most large crystallographic laboratories now have robotics systems, and the most sophisticated also automate the visualization of the crystallization experiments, to monitor the appearance of crystalline material. Such developments [e.g., Ref. 1] are adding computer visualization and pattern recognition to the informatics requirements. 

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... 

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