Isoelectric focusing electrophoresis scale

There are three distinct modes of electrophoresis zone electrophoresis, isoelectric focusing, and isotachophoresis. These three methods may be used alone or in combination to separate molecules on both an analytical (p.L of a mixture separated) and preparative (mL of a mixture separated) scale. Separations in these three modes are based on different physical properties of the molecules in the mixture, making at least three different analyses possible on the same mixture. 

Among the electrophoretic methods of chiral resolution, various forms of capillary electrophoresis such as capillary zone electrophoresis (CZE), capillary isotachophoresis (CIF), capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), affinity capillary electrophoresis (ACE), and separation on microchips have been used. However, in contrast to others, the CZE model has been used frequently for this purpose [44]. On the other hand, drawbacks associated with the electrophoretic technique due to lack of development of modem chiral phases have limited the application of these methods. Moreover, the electrophoretic techniques cannot be used at the preparative scale, which represents an urgent need of chiral separation science. 

P. Schmitt-Kopplin, Capillary Electrophoresis Methods and Protocols, Humana, 2007, ISBN 9781588295392 J.P. Landers, Handbook of Capillary Microchip Electrophoresis and Associated Microtechniques, CRC Press, Boca Raton, Florida, 2007, ISBN 9780849333293, C. Henry, Microchip Capillary Electrophoresis, Humana, 2006, ISBN 9781588292933.] The bands are eluted according to their isoelectric points. Isoelectric focusing standards are available which can be used in a preliminary run in order to calibrate the effluent from the column, or alternatively the pH of the effluent is recorded using a glass electrode designed for the purpose. Several efficient commercial equipment are available for separating proteins on a preparative and semi-preparative scale. 

Many methods for the preparation of pure serum albumin are available, and some can be scaled down from techniques used by blood transfusion services to process protein components from outdated blood. Assay can be by affinity chromatography (GlOa), HPLG, isoelectric focusing, mobile affinity electrophoresis, and thiobarbituric acid photometry (NIO). The last method employed acid (pH 1) hydrolysis for 8 hours at 115°C to liberate the... 

The most widely used methods by far employ coherent or granular gels. The former are most commonly polyacrylamide or agarose and the latter is usually Sephadex. Gels have been used in the preparative applications of each of the three common electrophoretic modes, isoelectric focusing (lEF), isotachophoresis (ITP) and zone electrophoresis (ZE). Regardless of the mode utilized, the procedure is most often a batch operation and employs either a cylindrical flat bed or annular separation chamber. Isotachophoresis has been applied to preparative scale fractionations in both polyacrylamide (.3) and Sephadex... 

Figure 1. A two-dimensional electrophoresis (2DE) separation of 80 tg of heart (ventricle) proteins. The first dimension comprised an 18 cm non-linear pH 3-10 immobihsed pH gradient (IPG) subjected to isoelectric focusing. The second dimension was a 21 cm 12% SDS-PAGE (sodium dodecylsulphate polyacrylamide gel electrophoresis) gel. Proteins were detected by silver staining. The non-linear pH range of the first-dimension IPG strip is indicated along the top of the gel, acidic pH to the left. The Mr (relative molecular mass) scale can be used to estimate the molecular weights of the separated proteins.

ID Separation of Proteins There has been considerable research devoted to tbe development of nricioflu-idic platforms capable of performing small-scale protein separations. In addition to diffusion based methods [3], nearly every type of electrokineticaUy driven separation has been demonstrated in a cbip-based platform, including free-flow electrophoresis, capillary electrophoresis, capillary gel electrophoresis, isoelectric focusing (lEF), micellar electrokinetic chromatography and capillary electrochemical chromatography. 

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