Gel Electrophoresis Studies

Hu, D. H., Kimura, S., and Maruyama, K. (1986). Sodium dodecyl sulfate gel electrophoresis studies of connectin-like high molecular weight proteins of various types of vertebrate and invertebrate muscles./. Biochem. (Tokyo) 99, 1485—1492. 

Jonsson, M. Akerman, B. Norden, B. Orientation of DNA during gel electrophoresis studied with linear dichroism spectroscopy. Biopolymers 1988 27 381-414. 

Akerman, A. Jonsson, M. Moore, D. Schellman, J. Conformational dynamics of DNA during gel electrophoresis studied by dichroism spectroscopy. Electrophoresis of Large DNA Molecules Theory and Applications. Cold Spring Harbor Laboratory Press, 1990 23. 

About 200 polypeptides can be resolved by gel electrophoresis studies of isolated axonemes. Analysis indicates at least 6 proteins in the spoke heads and 11 others in the arms of the spokes. Much of this apparatus seems to be directly involved in the beating motions of cilia and flagella. 

The genomes of Streptomyces bacteria are very large by bacterial standards. Pulsed-field gel electrophoresis studies have revealed that the genomes of... 

A. Parkinson, M. Hawken, M. Hall, K. J. Sanders, and A. Rodger, Amine induced Z-DNA in poly(dG-dC)center dot poly(dG-dC) Circular dichroism and gel electrophoresis study, Phys. Chem. Chem. Phys. 2(23), 5469-5478 (2000). 

Because of the diflBculties of separating the hydrophobic proteins of the inner membrane by gel electrophoresis, studies were more successful in which the product of mitochondrial protein synthesis was charae-terized using specific inhibitors. As a result the activity of a few mitochondrial enzymes seems to be linked to the function of the mitochondrial genome, or at least to the function of mitoribosomes. 

Gasiorowski, K., Brocos, B. DNA repair of hydrogen peroxide-induced damage in human lymphocytes in the presence of four antimutagens. A study with alkaline single cell gel electrophoresis (comet assay). Cellular Molecular Biology Letters, Vol.6, (2001), pp. 897-911, ISSN 1425-8153... 

Techniques for study of higher orders of protein stmcture include x-ray crystallography, NMR spectroscopy, analytical ultracentrifugation, gel filtration, and gel electrophoresis. 

The number of different proteins in a membrane varies from less than a dozen in the sarcoplasmic reticulum to over 100 in the plasma membrane. Most membrane proteins can be separated from one another using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), a technique that has revolutionized their study. In the absence of SDS, few membrane proteins would remain soluble during electrophoresis. Proteins are the major functional molecules of membranes and consist of enzymes, pumps and channels, structural components, antigens (eg, for histocompatibility), and receptors for various molecules. Because every membrane possesses a different complement of proteins, there is no such thing as a typical membrane structure. The enzymatic properties of several different membranes are shown in Table 41-2. 

Prokaryotic cells express hundreds to thousands of proteins while higher eukaryotes express thousands to tens of thousands of proteins at any given time. If these proteins are to be individually identified and characterized, they must be efficiently fractionated. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has typically been use to study protein mixtures of <100 proteins. Onedimensional electrophoresis is useful because nearly all proteins are soluble in SDS, molecules ranging from approximately 10,000 to 300,000 molecular weight can be resolved, and extremely basic or acidic proteins can be visualized. The major disadvantage to one-dimensional gels is that they are not suitable for complex mixtures such as proteins from whole cell lysates. 

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. 

Current proteomics studies rely almost exclusively on 2D gel electrophoresis to resolve proteins before MALDI-TOF or ESI-MS/MS approaches. A drawback of the 2D gel approach is that it is relatively slow and work intensive. In addition, the in-gel proteolytic digestion of spots followed by mass spectrometry is a one-at-a-time method that is not well suited for high throughput studies. Therefore, considerable effort is being directed towards alternate methods for higher throughput protein characterization. 

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