Isoelectric focusing mechanism

Several modes of capillary electrophoretic separation are available ordinary CE, capillary zone electrophoresis, capillary electrokinetic chromatography, capillary gel electrophoresis, capillary electrochromatography, capillary isota-chophoresis, and capillary isoelectric focusing. The different separation mechanisms make it possible to separate a wide variety of substances depending on their mass, charge, and chemical nature.53... 

The pectate lyase activities secreted by Clostridium populeti, isolated from a poplar based methane digestor (28), were compared with those secreted by E. chrysanthemi and L. multiparus. Isoelectric focusing followed by overlay and activity analysis revealed the presence of one or two activity bands with pi values from 4.5 to 4.7 for both the C. populeti and the L. multiparus. The kinetic depolymerization profiles of C. populeti also revealed an exolytic/endolytic mechanism, typified by the high ratio of trimer and/or dimer to hexamer in the early stages of the reaction (Table I). 

Capillary zone electrophoresis (CZE) is the most common electrophoretic separation technique due to its simplicity of operation and its flexibility. It is the standard mode for drug analysis, identification of impurities, and pharmacokinetic studies. Other separation modes, such as capillary isotachopho-resis (CITP), micellar electrokinetc chromatography (MEKC), capillary electrochromatography (CEC), capillary gel electrophoresis (CGE), capillary isoelectric focusing, and affinity capillary electrophoresis (ACE), have then-advantages in solving specific separation problems, since the separation mechanism of each mode is different. 

The second separation mechanism is found in capillary isoelectric focusing (cIEF), where analytes are separated on the basis of isoelectric points. The third mechanism is found in capillary isotachophoresis (cITP), where all of the solutes travel at the same velocity through the capillary but are... 

Isoelectric focusing was developed to separate proteins on the basis of differences in their pi values. The mechanism of IEF can be treated mathematically and the classical analysis is presented in the Appendix. On the other hand, the mechanism of IEF is relatively easy to conceptualize and lends itself to a simple description. The historical, theoretical, and practical aspects of IEF are well documented. Many experimental details can be found in several monographs and review articles.1,2,8-17 Refs. 1 and 2 are particularly recommended. They provide valuable insights from one of the pioneers in the field. 

Thormann, W., Mosher, R. A., and Bier, M. (1986). Experimental and theoretical dynamics of isoelectric focusing Elucidation of a general separation mechanism. J. Chromatogr. 351, 17-29. 

Fibroblasts from these patients express the normal number of surface LDL receptors which exhibit normal binding capacity. Moreover, immunodetection of LDL receptors analyzed by two-dimensional isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that internalization-defective receptors are indistinguishable from those of normal cells [105]. Despite the presence of the normal number of intact receptors, fibroblasts from these patients are unable to take up LDL by receptor-mediated endocytosis. The mutation thus appears to be in some aspect of the receptor internalization mechanism [87]. Clinical symptoms resemble those of receptor-negative or receptor-defective FH. 

Electrophoretic modes include zone, gel sieving, isoelectric focusing, and isotachophoresis. But electrophoresis has a distinct advantage over HPLC for analysis of biopolymers a vastly superior resolving power, especially in a two-dimensional format, where two separation mechanisms can be used in succession. 

The terms cellulolytic enzymes and cellulases now enjoy a workable definition. This has been made possible through the separation of the components of the cellulolytic complex, as well as some clarification of their mechanism of action. New viscometric, isoelectric focusing, gel filtration, and other techniques for the determination of the enzymic components have been devised. Detailed observations on the molecular sites of hydrolytic reactions, the susceptibility of the enzymic reactions to metallic ions, proteolytic enzymes, and other chemicals also provided considerable insight into the nature of cellulase. 

Figure 8.9. Schematic diagram of the two-step process in capillary isoelectric focusing using a coated capillary column. The top figure illustrates the steady state focussing mechanism and the bottom figure chemical mobilization by adding salt to the cathode reservoir. The focussed protein bands are transported past the detector located at the cathode end of the capillary.

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