Isoelectric focusing introduction

This introduction was intended to merely provide basic concepts and principles of electrophoretic separation. It should be noted that a wide variety of electrophoretic techniques, such as isoelectric focusing and two-dimensional gel... 

A second example of the improvements in isoelectric focusing generated by the introduction of IPG gels is shown in Figure 11.8, where protein samples obtained from bean seeds are subjected to IEF for up to 12 h, using carrier ampholytes and immobilized pH gradients.11 In principle, proteins focused at their isoelectric pH values are not subject to changes in position with time, since IEF is a steady... 

While some techniques require the absence of electroosmotic flow during the separation itself (capillary gel electrophoresis and capillary isoelectric focusing), most common techniques exploit electroosmotic flow for sample introduction and detection. Electrophoresis in buffer-filled capillaries uses electroosmotic flow in an analogous manner to a chromatographic mobile phase the flow is used to transport analyte from cathode to anode and separation occurs continuously between introduction and detection. 

The introduction of density gradients allowed several improvements in the analytical and preparative capabilities of centrifugal processes. First, it is possible to pour the sample in the top of a tube, in a more or less thin layer differences in density between sample and separation medium avoids convection and delays the homogenization process. Second, it is possible to diminish convection fluid movements in the tube convection can be produced by a difference in temperature between different parts of the centrifugal rotor. Third, it is possible to focus a particle when its reaches its own density, in a process analogous to isoelectric focusing. 

Methods for AMY isoenzymes based on electrophoresis, ion-exchange chromatography, isoelectric focusing, selective inhibition of the S-AMY by a wheat germ inhibitor, immunoprecipitation by a monoclonal antibody, and immunoinhibition have been introduced. However, only the methods based on the selective isoenzyme inhibition by monoclonal antibodies have shown sufficient precision, reliability, practicability, and analytical speed to allow the introduction of the P-AMY determination in clinical practice. 

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