Two-dimensional electrophoretic

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. 

He, Q Johnson, CS, Two-Dimensional Electrophoretic NMR for the Measurement of Mobilities and Diffusion in Mixtures, Journal of Magnetic Resonance 81, 435, 1989. 

Shadpour, H., Soper, S.A. (2006). Two-dimensional electrophoretic separation of proteins using poly(methyl methacrylate) microchips. Anal. Chem. 78, 3519-3527. 

Nilsson CL et al. Identification of protein vaccine candidates from Helicobacter pylori using a preparative two-dimensional electrophoretic procedure and mass spectrometry. Anal Chem 2000 72 2148-2153. 

Norbeck J et al. Two-dimensional electrophoretic separation of yeast proteins using a non-linear wide range (pH 3-10) immobilized pH gradient in the first dimension reproducibility and evidence for isoelectric focusing of alkaline (pi >7) proteins. Yeast 1997 13 1519-1534. 

One concern relating to SDS-PAGE-based purity analysis is that contaminants of the same molecular mass as the product will go undetected as they will co-migrate with it. Two-dimensional electrophoretic analysis would overcome this eventuality in most instances. 

Two-dimensional electrophoresis is normally run so that proteins are separated from each other on the basis of a different molecular property in each dimension. The most commonly utilized method entails separation of proteins by isoelectric focusing (see below) in the first dimension, with separation in the second dimension being undertaken in the presence of SDS, thus promoting band separation on the basis of protein size. Modified electrophoresis equipment that renders two-dimensional electrophoretic separation routine is freely available. Application of biopharmaceuti-cal finished products to such systems allows rigorous analysis of purity. 

WAGONER, W LOSCHKE, D.C., HADWIGER, L.A., Two-dimensional electrophoretic analysis of in vivo and in vitro synthesis of proteins in peas inoculated with compatible and incompatible Fusarium solani, Physiological Plant Path., 1982,20,99-107. 

Shadpour, H. Soper, S. A. Two-Dimensional Electrophoretic Separation of Proteins using Poly(methyl methacrylate) Microchips. Anal. Chan. 20 06, 78, 3519-35 27. 

Young DA, Voris BP, Maytin EV, Colbert BA. (1983) Very-high-resolution two-dimensional electrophoretic separation of proteins on giant gels. MethodsEnzymol9, 190-214. 

Mass spectrometry provides a wealth of information for proteomics research, enzymology, and protein chemistry in general. The techniques require only miniscule amounts of sample, so they can be readily applied to the small amounts of protein that can be extracted from a two-dimensional electrophoretic gel. The accurately measured molecular mass of a protein is one of the critical parameters in its identification. Once the mass of a protein is accurately known, mass spectrometry is a convenient and accurate method for detecting changes in mass due to the presence of bound cofactors, bound metal ions, covalent modifications, and so on. 

Microsequencers permit sequence analysis on minute amounts of protein. Microsequencing can be used in conjunction with two-dimensional electrophoretic separations of proteins such as that shown in Box 3-C. The proteins in the polyacrylamide gel are electropho-retically transferred onto a porous sheet (membrane) of an inert material such as polyvinyl difluoride.249-251 After staining, a selected spot is cut out and placed into the sequencer. To avoid the problems associated with blocked N termini, the protein may be treated with proteases on the membrane and the resulting peptide fragments may then be separated on a narrow-bore HPLC column and sequenced.240... 

L Bini, B Magi, B Marzocchi, C Cellesi, B Berti, R Raggiaschi, A Rossolini, V Pallini. Two-dimensional electrophoretic patterns of acute-phase human serum proteins in the course of bacterial and viral diseases. Electrophoresis 17 612-616,1996. 

FA Witzmann, CD Fultz, JC Lipscomb. Toxicant-induced alterations in two-dimensional electrophoretic patterns of hepatic and renal stress proteins. Electrophoresis 17 198-202, 1996. 

Witzmann FA, Daggett DA, Fultz CD, Nelson SA, Wright LS, Kornguth SE, Siegel FL. Glutathione S-transferases two-dimensional electrophoretic protein markers of lead exposure. Electrophoresis 1998 19 1332-1335. 

Witzmann FA, Fultz CD, Mangipudy RS, Mehendale HM. Two-dimensional electrophoretic analysis of compartment-specific hepatic protein charge modification induced by thioacetamide exposure in rats. Fundam Appl Toxicol 1996 31 124-132. 

The reagents in current use for protein crosslinking have one chemically reactive and one photochemically reactive arm, often connected by a cleavable bridge for subsequent two-dimensional electrophoretic analyses. Chemical attachment is carried out first, and the crosslink is completed by photolysis. Many of these so-called heterobifunctional molecules are listed in Table 5.1 and some of them, conforming to the criteria outlined below,... 

While the two-dimensional electrophoretic analysis described above is suitable for biological complexes that contain only a few major components (such as the human erythrocyte membrane), it is not feasible to extend its use to more complex systems such as the plasma membranes of many other cell types. Nevertheless, several ways to improve the method may prove useful. Immunoprecipitation of all the crosslinking products... 

Atkinson, B. G. Podesta, R. B. (1982). Two-dimensional electrophoretic separation and fluorographic analysis of the gene products synthesised by Hymenolepis diminuta with particular reference to the parasite-specific polypeptides in the brush border membrane. Molecular and Biochemical Parasitology, 6 33-43. 

Developmental regulation of protein synthesis in Hymenolepis diminuta two-dimensional electrophoretic and fluorographic analysis of protein synthesis in oncospheres. Journal of... 

Robinson, J. C., B. S. Blumberg, and J. E. Pierce Studies on inherited variants of blood proteins. I. Two dimensional electrophoretic analysis of chromatographic fractions of serum proteins. J. Lab. Clin. Med. 60, 468 (1962). 

Xu, B., etal. (2005). Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database. Proteomics 5, 1083-1096. 

Stastny,]., Robertson, A. L, Fosslien, E. (1986). Basic proteins in the human aortic infima nonequilibrium two-dimensional electrophoretic analysis of tissue extracts. Exp. Mol. Pathol. 45, 279-286. 

Mathias, P. I., Fultz, C., Witzmann, F. (1997). Two-dimensional electrophoretic analysis of myocardial proteins from lead-exposed rabbits. Electrophoresis 18, 2978-2982. 

High-Resolution Two-Dimensional Electrophoretic Techniques 4.1. Introduction... 

Two-Dimensional Electrophoretic Analysis of Tissues and Body Fluids... 

A13. Anderson, N. G., Anderson, N. L., Tollaksen, S. L., Hahn, H., Giere, F., and Edwards, J., Analytical techniques for cell fractions. XXV. Concentration and two-dimensional electrophoretic analysis of human urinary proteins. Anal. Biochem. 95,... 

W5. Willard, K. E., and Anderson, N. L., Alterations of two-dimensional electrophoretic maps in human peripheral blood lymphocytes induced by concanavalin A. In Electrophoresis 79 (B. J. Badola, ed.), pp. 415-424. Walter de Gruyter, Berlin, 1980. 

Clark and Kricka have reviewed High-Resolution Analytical Techniques for Proteins and Peptides and Their Applications in Clinical Chemistry and include consideration of isotachophoresis, high-performance liquid chromatography, and high-resolution two-dimensional electrophoretic techniques for separation and analysis of complex protein mixtures. These techniques are not now widely used in clinical chemistry laboratories but represent the tools of the future, when laboratories will be required to measure gene products and the myriad proteins present, as in complex biologic fluids of significance in health and diseases. 

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