Carrier ampholyte isoelectric focusing

Figure 5. Two sucrose density gradient electrofocusing runs identical except for the polarity of the electrodes. The first, A, has been focused with the anode in the dense solution, i.e., at the bottom of the column, and the second, B, with the cathode in the dense solution. The pH-gradient is hardly affected by the superimposed sucrose density gradient. In the conductivity, however, there is a difference in the two experiments. With the cathode in the bottom, the conductivity lowering effect from the sucrose density gradient will add to the relatively low conductivity of the carrier ampholytes isoelectric near pH 9. In both curves one can observe the low conductivity of the carrier ampholytes around pH 7.

Rabilloud, T., Valette, C., and Lawrence, J. J. (1994). Two-dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte-pH gradients. Electrophoresis 15, 1552-1558. 

Wallevik, K. 1973. Isoelectric focusing of bovine serum albumin. Influence of binding of carrier ampholyte. Biochim. Biophys. Acta 322, 75-87. 

Govi, M., Bonoretti, G., Ciavata, C., and Sequi, P. (1994). Characterization of soil organic matter using isoelectric focusing a comparison of six commercial carrier ampholytes. Soil Sci. 157, 91-96. 

Shang TQ, Ginter JM, Johnston MV, Larsen BS, McEwen CN. Carrier ampholyte-free solution isoelectric focusing as a prefractionation method for the proteomic analysis of complex protein mixtures. Electrophoresis 2003 24 2359-2368. 

Shimao, K. (1987). Mathematical simulation of steady state isoelectric focusing of proteins using carrier ampholytes. Electrophoresis 8, 14-19. 

To remove carrier ampholytes when electrospray ionisation mass spectrometry is used on-line with capillary isoelectric focusing, a specially designed free-flow electrophoresis device can be coupled to the CIEF system [67],... 

Isoelectric focusing is a well documented technique that uses carrier ampholytes (Rilbe, 1973 Fawcett, 1975). A pH gradient can also be created using a thermal gradient in ordinary buffers (Luner and... 

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... 

Figure 11.8. Comparison of (a) carrier ampholyte (pH 4-6 range) and (b) IPG (pH 4—8 range) isoelectric focusing pattern stability over time, for the separation of a mixture of bean seed proteins.11 [Reprinted, with permission, from Methodology of two-dimensional electrophoresis with immobilized pH gradients for the analysis of cell lysates and tissue proteins , A. Gorg, W. Postel, A. Domscheit, and S. Gunther, in Two-Dimensional Electrophoresis , Proceedings of the International Two-Dimensional Electrophoresis Conference, Vienna, November 1988. A. T. Endler and S. Hanash, Eds., VCH Publishers, Weinheim, Germany, 1989. 1988 by VCH Verlagsgesellschaft mbH.]...

Figure 12.13. CIEF of cytochrome c (peak 1, pi 9.6), chymotrypsinogen A (peak 2, pi 9.0) and myoglobin (peaks 3 and 4, pi 7.2, and 6.8) on a 75-pm i. d. uncoated capillary, 60 cm long and 40 cm from anode to detector. Anolyte 10-mM phosphoric acid catholyte 20-mM NaOH. Running buffer contains 0.1% methylcellulose, 1% TEMED, and 5% Pharmalyte 3-10 carrier ampholytes. The UV 280-nm detection.11 [Reprinted, with permission, from J. R. Mazzeo and I. S. Krull, Anal. Chem. 63 (No. 24), 1991, 2852-2857. Capillary Isoelectric Focusing of Proteins in Uncoated Fused-Silica Capillaries Using Polymeric Additives . 1991 by American Chemical Society.]...

For isoelectric focusing (IFF) (see later section), a power supply that provides constant power is advisable. During electrophoresis, current drops significantly because of lower conductivity as carrier ampholytes focus at their isoelectric points and because of creation of zones of pure water. If a constant-voltage supply is used, frequent voltage adjustments may be necessary. Constant-current power supplies are not customarily used in lEF. Pulsed-power or pulsed-field techniques (see later section) require a power supply that can periodically change the orientation of the applied field relative to the direction of migration. 

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