Hostname: page-component-77c89778f8-cnmwb Total loading time: 0 Render date: 2024-07-17T08:00:27.115Z Has data issue: false hasContentIssue false
  • English
  • Français

Two dimensional electrophoresis and immunoblotting for the study of ovine whey protein polymorphism

Published online by Cambridge University Press:  01 June 2009

Gloria López-Gálvez ,
Manuela Juárez  and
Mercedes Ramos
Gloria López-Gálvez
Affiliation:
Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, España
Manuela Juárez
Affiliation:
Institute del Frío (CSIC), Cuidad Universitaria, 28040 Madrid, España
Mercedes Ramos
Affiliation:
Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, España
Get access Rights & Permissions [Opens in a new window]

Summary

Different electrophoretic techniques have been applied to the study of ovine whey protein polymorphism. Isoelectric focusing (IEF) over the pH gradient 3·5–9·5 was a suitable method for resolving both genetic variants of ovine β-lactoglobulin (β-lg); using this type of IEF, ovine β-lg A seemed to be defined by a major and a minor ‘satellite’ band. Approximate isoelectric point, relative molecular mass, amino acid composition and N-terminal sequence of this minor band were determined. A good separation of ovine β-lg was achieved using ultrathin layer isoelectric focusing (UTLIEF) over the pH gradient 2·5–7·0. However, addition of urea in UTLIEF gels led to some differences in the patterns of the ovine whey proteins when compared with those obtained by IEF on gels not containing urea. Two dimensional electrophoretic techniques provided a considerable separation of ovine whey proteins. Immunoblotting applied to a two dimensional separation permitted the identification of the bands belonging to β-lg and α-lactalbumin fractions.

Type
Original Articles
Information
Journal of Dairy Research , Volume 62 , Issue 2 , May 1995 , pp. 311 - 320
Copyright
Copyright © Proprietors of Journal of Dairy Research 1995

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Addeo, F., Moio, L., Chianese, L. & Di Luccia, A. 1989 [Detection of bovine milk in ovine milk or cheese by gel isoelectric focusing of β-lactoglobulin: applications and limitations.] Italian Journal of Food Science 1 45–52Google Scholar
Amigo, L. 1989 [Study of the Protein Fraction of Milk from Ewes, Goats and Cows. Analytical Criteria for Determining Mixtures of Milk in Cheeses.) PhD thesis, Universidad Autónoma de MadridGoogle Scholar
Aschaffenburg, R. & Drewry, J. 1959 New procedure for the routine determination of the various non-casein proteins of milk. 15th International Dairy Congress, London 3 1631–1637Google Scholar
Bell, K. & McKbnzie, H. A. 1964 β-Lactoglobulins. Nature 204 1275–1279CrossRefGoogle ScholarPubMed
Bell, K. & McKenzie, H. A. 1967 The whey proteins of ovine milk. β-Lactoglobulins A and B. Biochimica et Biophysica Acta 147 123–134CrossRefGoogle ScholarPubMed
Cerning-Beroard, J. & Zevaco, C. 1984 Purification and characterization of porcine κ-casein. Journal of Dairy Research 51 259–266Google Scholar
Chianese, L., Garro, G., Addeo, F., López-Gálvez, G. & Ramos, M. 1993 Discovery of an ovine αs2-casein variant. Journal of Dairy Research 60 485–493CrossRefGoogle Scholar
Chianese, L., Mauriello, R., Moio, L., Intorcia, N. & Addeo, F. 1992 Determination of ovine casein heterogeneity using gel electrophoresis and immunochemical techniques. Journal of Dairy Research 59 39–47CrossRefGoogle ScholarPubMed
Chiofalo, L. & Micari, P. 1987 [Present knowledge of milk protein variants in ovine populations bred in Sicily. Experimental observations.] Scienza e Tecnica Lattiero-Casearia 38 104–114Google Scholar
Erhardt, G. 1989 Evidence for a third allele at the β-lactoglobulin (β-lg) locus of sheep milk and its occurrence in different breeds. Animal Genetics 20 197–204CrossRefGoogle ScholarPubMed
Erhardt, G., Godovac-Zimmermann, J. & Conti, A. 1989 Isolation and complete primary sequence of a new ovine wild-type β-lactoglobulin C. Biological Chemistry Hoppe-Seyler 370 757–762CrossRefGoogle ScholarPubMed
Fernández-Esplá, M. D., López-Gálvez, G. & Ramos, M. 1993 Isolation of ovine β-lactoglobulin genetic variants by chromatofocusing: heterogeneity of β-lactoglobulin A. Chromatographia 37 43–46Google Scholar
Fox, K. K., Holsinger, V. H., Posati, L. P. & Pallansch, M. J. 1967 Separation of β-lactoglobulin from other milk serum proteins by trichloroacetic acid. Journal of Dairy Science 50 1363–1367Google Scholar
Gave, P., Hue-Delahaie, D., Mercier, J. -C., Soulier, S., Vilotte, J. -L. & Furet, J. -P. 1986 Ovine β- lactoglobulin messenger RNA: nucleotide sequence and mRNA levels during functional differentiation of the mammary gland. Biochimie 68 1097–1107Google Scholar
Glrl, L. 1990 Chromatofocusing. Methods in Enzymology 182 380–392Google Scholar
Grosclaude, F. 1988 [Genetic polymorphism in the main milk proteins in cattle. Relationship with milk yield, composition and suitability for cheese production.] INRA Productions Animates 1 5–17, 75Google Scholar
Hillier, R. M. 1976 The quantitative measurement of whey proteins using polyacrylamide-gel electrophoresis. Journal of Dairy Research 43 259–265Google Scholar
King, J. W. B. 1969 The distribution of sheep β-lactoglobulins. Animal Production 11 53–57Google Scholar
Krause, I. 1991 Modification of the Reference Method for the detection of milk whey proteins in ewe's milk cheese. Working Paper Introduced by the German Delegation in the Meeting of the Expert Group of Chemists. Brussels, April 12thGoogle Scholar
Liberatori, J. 1977 β-Lactoglobulins. Chemical and structural studies. Folia Veterinaria Latino. 7 205–222Google Scholar
López-Gálvez, G., Amigo, L., Ramos, M. & Juarez, M. 1991 [Study of ovine milk whey proteins by electrophoretic techniques.] In XX Reunión Científica del Grupo de Cromalografía y Técnicas Afines, San Sebastian, SpainGoogle Scholar
Mao, F. C. & Bremel, R. D. 1991 Enzyme-linked immunosorbent assays for bovine α-lactalbumin and β- lactoglobulin in serum and tissue culture media. Journal of Dairy Science 74 2946–2951Google Scholar
Morr, C. V. 1967 Effect of urea upon the physical properties of β-lactoglobulins A and B. Journal of Dairy Science 50 1752–1759Google Scholar
Ruíz, E. & Santillana, I. 1986 [Determination of cows', goats' and ewes' mixture milks by polyacrylamide gel isoelectric focusing.] Alimentaria 171 55–64Google Scholar
Schmidt, D. V. & Ebner, K. E. 1972 Multiple forms of pig, sheep and goat α-laetalbumin. Biochimica et Biophysica Acta 263 714–720CrossRefGoogle Scholar
Seibert, B., Erhardt, G. & Senft, B. 1985 Procedure for simultaneous phenotyping of genetic variants in cow's milk by isoelectric focusing. Animal Blood Groups and Biochemical Genetics 16 183–191CrossRefGoogle ScholarPubMed
Trieu-Cuot, P. & Gripon, J.-C. 1981 Electrofocusing and two-dimensional electrophoresis of bovine caseins. Journal of Dairy Research 48 303–310Google Scholar