Abstract
Electrophoresis, a technique consisting of the migration of electrically charged molecules under an applied electric field, occupies one of the most important places in the history of the study of hemoglobin (Hb). HbS, the first abnormal Hb described, was discovered in 1949 by Pauling et al. (1), using moving boundary electrophoresis. Later, Hb variants were detected by zone electrophoresis on paper, starch gel, or cellulose acetate (2,3). With the exception of cellulose acetate electrophoresis, which is still used in some laboratories, these procedures have been replaced by isoelectric focusing (IEF) (4). In IEF, a pH gradient is established by carrier ampholytes subjected to an electric current. The Hb molecule migrates across this gradient until it reaches the position where its net charge is zero (isoelectric point [pI]). It then concentrates into a sharp band.
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References
Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C. (1949) Sickle cell anemia, a molecular disease. Science 110, 543.
Huisman, T. H. J. and Jonxis, J. H. P. (1977) The Hemoglobinopathies Techniques of Identification, Clinical and Biochemical Analysis, vol. 6, Marcel Dekker, New York.
Huisman, T. H. J. (1986) Introduction and review of standard methodology for the detection of hemoglobin abnormalities, in The Hemoglobinopathies, Methods in Hematology, vol. 15 (Huisman, T. H. J., ed.), Churchill Livingstone, Edinburgh.
Righetti, P. G., Gianaza, E., Bianchi-Bosisio, A., and Cossu, G. (1986) Conventional isoelectric focusing and immobilized pH gradients for hemoglobin separation and identification, in The Hemoglobinopathies, Methods in Hematology, vol. 15 (Huisman, T. H. J., ed.), Churchill Livingstone, Edinburgh, p. 47.
Barwick, R. C. and Schneider, R. G. (1980–1981) The computer-assisted differentiation of hemoglobin variants, in Human Hemoglobins and Hemoglobinopathies: A Review to 1981. Texas Reports on Biology and Medicine, vol 40. (Schneider, R. G., Charache, S., and Schroeder, W. A., eds.), University of Texas Medical Branch, Galveston, TX, pp. 143–156.
Basset, P., Beuzard, Y., Garrel, M. C., and Rosa, J. (1978) Isoelectric focusing of human hemoglobin: its application to screening, to the characterization of 70 variants, and to the study of modified fractions of normal hemoglobins. Blood 51, 971–982.
Righetti, P. G. (1983) Isoelectric Focusing: Theory, Methodology and Applications, Elsevier, Amsterdam.
Bjellqvist, B., Ek, K., Righetti, P. G., Gianazza, E., Gorg, A., Westermeier, R., and Postel, W. (1982) Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J. Biochem. Biophys. Methods 6, 317–339.
Righetti, P. G., Gianazza, E., Bianchi-Bosisio, A., Wajcman, H., and Cossu, G. (1989) Electrophoretically silent hemoglobin mutants as revealed by isoelectric focusing in immobilized pH gradients. Electrophoresis 10, 595–599
Cossu, G., Manca, M., Pirastru, M. G., Bullitta, R., Bianchi-Bosisio, A., Gianazza, E., and Righetti, P. G. (1982) Neonatal screening of β-thalassemias by thin layer isoelectric focusing. Am. J. Hematol. 13, 149–157.
Drysdale, J. W., Righetti, P., and Bunn, H. F. (1971) The separation of human and animal hemoglobins by isoelectric focusing in polyacrylamide gel. Biochim. Biophys. Acta. 229, 42–50.
Schneider, R. G. and Barwick, R. C. (1982) Hemoglobin mobility in citrate agar electrophoresis: its relationship to anion binding. Hemoglobin 6, 199–208.
Alter, B. P., Goff, S. C., Efremov, G. D., Gravely, M. E., and Huisman, T. H. J. (1980) Globin chain electrophoresing: a new approach to the determination of the G/A ratio in fetal haemoglobin and to the studies of globin synthesis Br. J. Haematol. 44, 527–534.
Radola, B. J. (1973) Analytical and preparative isoelectric focusing in gel-stabilized layers. N Y Acad. Sci. 209, 127–143.
Westermeier, R. (1997) Electrophoresis in Practice, 2nd ed., VCH Verlagsgesellschaft, Weinheim, Germany.
Harano, T., Ueda, S., Harano, K., and Shibata, S. (1980) Improved method for quantitation of biosynthesized human globin chains in reticulocytes by use of urea cellulose acetate membrane electrophoresis. Proc. Jap. Acad. 56(B), 230–234.
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Wajcman, H. (2003). Electrophoretic Methods for Study of Hemoglobins. In: Nagel, R.L. (eds) Hemoglobin Disorders. Methods in Molecular Biology™, vol 82. Humana Press. https://doi.org/10.1385/1-59259-373-9:093
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DOI: https://doi.org/10.1385/1-59259-373-9:093
Publisher Name: Humana Press
Print ISBN: 978-0-89603-962-9
Online ISBN: 978-1-59259-373-6
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