Focusing of Proteins

Another important and effective use of electrophoresis for the analysis of proteins is isoelectric focusing (IEF), which examines electrophoretic mobility as a function of pH. The net charge on a protein is pH dependent. Proteins below their isoelectric pH (pHj, or the pH at which they have zero net charge) are positively charged and migrate in a medium of fixed pH toward the negatively charged cathode. At a pH above its isoelectric point, a 

Since different protein molecules in mixtures have different pH, values, it is possible to use IEF to separate proteins. In addition, the pH, of each protein in the mixture can be determined by measuring the pH of the region where the protein is focused. 

The protein sample can be loaded on the gel in either of two ways. A concentrated, salt-free sample can be layered on top of the gel as previously described for ordinary gel electrophoresis. Alternatively, the protein can be added directly to the gel preparation, resulting in an even distribution of 

---

Righetti, P. G., Isoelectric focusing of proteins in conventional and immobilized pH gradients, in Protein Structure A Practical Approach, Creighton, T. E., Ed., IRL Press, New York, 1989, 23. 

Shen, Y., Berger, S.J., Smith, R.D. (2001). High-efficiency capillary isoelectric focusing of protein complexes from Escherichia coli cytosolic extracts. J. Chromatogr. A 914,257-264. 

Allen RC, Saravis CA, Maurer HR (1984) Gel electrophoresis and isoelectric focusing of proteins. Selected techniques. W. de Gruyter, Berlin Chrambach A, Dunn MJ, Radola BJ (eds.) (1987 foil.) Advances in electrophoresis, vol. 1 foil., VCH, Weinheim Hames BD, RickwoodD (eds.) (1990) Gel electrophoresis of proteins a practical approach, 2nd ed., Oxford University Press, New York Deyl Z, Chrambach A, Everaerts FM, PrusikZ (eds.) (1983) Electrophoresis. A survey of techniques and applications. J Chromatogr Library, vol. 18B, Elsevier, Amsterdam... 

Salaman, M. R. and Williamson, A. R. 1971. Isoelectric focusing of proteins in the native and denatured states. Anomalous behavior of plasma albumin. Biochem. J. 122, 93-99. 

The apoE phenotype may be assigned after isoelectric focusing of proteins obtained by delipidating VLDL, obtained either by ultracentrifugation (H19, P2, U5, W4, W6) or heparin-Mn2+ precipitation (U4) of serum. The assignment of a phenotype is usually straightforward (H13) if not, the elimination of sialylated components with neuraminidase may be helpful (H13). 

Mazzeo, J., and Krull, I. (1991). Capillary isoelectric focusing of proteins in uncoated fused-silica capillaries using polymeric additives. Anal. Chem. 63, 2852-2857. 

Bier, M., and Marquez, R. B. (1994). High-resolution large-scale isoelectric focusing of proteins in a chemically defined buffer system. Tech. Protein Chem. 5, 249-258. 

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

Pressure denaturation of protein has been one of the problems in the focus of protein research due not only to its significance in science [47-49], but also to its importance in industrial applications, including food processing [50], The molecular mechanism of the process has not been clarified for a long time, especially concerning the role played by water or hydration. We have applied the RISM/3D-RISM theory to this problem to clarify the molecular mechanism behind the thermodynamics process [51]. 

Isoelectric focusing of proteins in mixed solvents in the range of normal and subzero temperatures is a very promising method. It... 

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.]...

Courtesy - R.C. Allen, C.A. Saravis, H.R. Maurer, Gel Electrophoresis and Isoelectric Focusing of Proteins, de Gruyter, Berlin, Ger 1984)... 

Allen, R. C., Saravis, C. A., Maurer, H. R., "Gel Electrophoresis and Isoelectric Focusing of Proteins Selected Techniques", Walter de Gruyter, Berlin, 1984. 

FIGURE 30-16 Capillary isoelectric focusing of proteins. Isoeleciric pi listed atx)ve the peaks. Detection was by UV absorption. (From T. Wehr. M. Zhu, R. Rodriguez. O Burke, and K. Duncan. Amen Biotech, Lab., 1990,6.22. With permission.)... 

Pulsed Field Gel Electrophoresis (PFGE) Isoelectric Focusing of Proteins 1 27... 

Thormaim, W., Huang, T., Pawliszyn, J., and Mosher, R.A., High-resolution computer simulation of the dynamics of isoelectric focusing of proteins, Electrophoresis, 25, 324, 2004. 

Shimao, K., Computer simulation of electrophoretic transport. II. Modified general procedure and application to isoelectric focusing of proteins using immobilised pH gradient, Jap. J. Electrophor., 38, 221, 1994. 

Tracy, N. I. and Ivory, C. F., Preparative isoelectric focusing of proteins using binary buffers in a vortex-stabilized, free-flow apparatus. Electmphoresis 2004, 25,1748-1757. 

Humble, P. H., Kelly, R. T., Woolley, A. T., Tolley, H. D., and Lee, M. L., Electric field gradient focusing of proteins based on shaped ionically conductive acrylic polymer, Anal. Chem., 76, 5641, 2004. 

Figure 9. Easily assembled apparatus for density gradient isoelectric focusing of proteins. A narrow glass tube is inserted into a glass cylinder within this a platinum wire extends almost to the bottom. The other electrode is a loop of platinum wire at the surface of the liquid. (Svensson, 3.)...

O. Vesterberg, Karolinska Institute , Stockholm, Inaugural Dissertation. Isoelectric Focusing of Proteins (1968b). 

Ivory CF, Huang Z (1999) Dynamic field-gradient focusing of proteins. Abstr Pap Am Chem Soc 217 U876-U876... 

Humble PH et al (2004) Electric field gradient focusing of proteins based on shaped ionically conductive acrylic polymer. Anal Chem 76(19) 5641-5648... 

Pawliszyn J, Wu J (1993) Ampholyte-free isoelectric focusing of proteins in cone-shaped capillaries. J Mitmcolnmn 5 397... 

Righetti PG, Gelfi C, and Gonti M (1997) Current trends in capillary isoelectric focusing of proteins. Journal of Chromatography B 699 91-104. 

Agarose gels for zone electrophoresis or isoelectric focusing of protein samples are mostly applied in the area of clinical diagnostics. For this purpose the gels are cast on horizontal glass plates or plastic films as thin layers of about 1 mm. 

---