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. 1985 Aug 1;229(3):785–790. doi: 10.1042/bj2290785

Detection of the low-density-lipoprotein receptor with biotin-low-density lipoprotein. A rapid new method for ligand blotting.

D P Wade, B L Knight, A K Soutar
PMCID: PMC1145125  PMID: 4052026

Abstract

A new technique has been developed to identify low-density-lipoprotein (LDL) receptors on nitrocellulose membranes, after transfer from SDS/polyacrylamide gels, by ligand blotting with biotin-modified LDL. Modification with biotin hydrazide of periodate-oxidized lipoprotein sugar residues does not affect the ability of the lipoprotein to bind to the LDL receptor. Bound lipoprotein is detected with high sensitivity by a streptavidin-biotin-peroxidase complex, and thus this method eliminates the need for specific antibodies directed against the ligand. The density of the bands obtained is proportional to the amount of pure LDL receptor protein applied to the SDS/polyacrylamide gel, so that it is possible to quantify LDL receptor protein in cell extracts. Biotin can be attached to other lipoproteins, for example very-low-density lipoproteins with beta-mobility, and thus the method will be useful in the identification and isolation of other lipoprotein receptors.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beisiegel U., Schneider W. J., Brown M. S., Goldstein J. L. Immunoblot analysis of low density lipoprotein receptors in fibroblasts from subjects with familial hypercholesterolemia. J Biol Chem. 1982 Nov 10;257(21):13150–13156. [PubMed] [Google Scholar]
  2. Bilheimer D. W., Goldstein J. L., Grundy S. M., Brown M. S. Reduction in cholesterol and low density lipoprotein synthesis after portacaval shunt surgery in a patient with homozygous familial hypercholesterolemia. J Clin Invest. 1975 Dec;56(6):1420–1430. doi: 10.1172/JCI108223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chung B. H., Wilkinson T., Geer J. C., Segrest J. P. Preparative and quantitative isolation of plasma lipoproteins: rapid, single discontinuous density gradient ultracentrifugation in a vertical rotor. J Lipid Res. 1980 Mar;21(3):284–291. [PubMed] [Google Scholar]
  4. Daniel T. O., Schneider W. J., Goldstein J. L., Brown M. S. Visualization of lipoprotein receptors by ligand blotting. J Biol Chem. 1983 Apr 10;258(7):4606–4611. [PubMed] [Google Scholar]
  5. Dresel H. A., Otto I., Weigel H., Schettler G., Via D. P. A simple and rapid anti-ligand enzyme immunoassay for visualization of low-density lipoprotein membrane receptors. Biochim Biophys Acta. 1984 Oct 4;795(3):452–457. doi: 10.1016/0005-2760(84)90172-3. [DOI] [PubMed] [Google Scholar]
  6. Gahmberg C. G., Andersson L. C. Selective radioactive labeling of cell surface sialoglycoproteins by periodate-tritiated borohydride. J Biol Chem. 1977 Aug 25;252(16):5888–5894. [PubMed] [Google Scholar]
  7. Guesdon J. L., Ternynck T., Avrameas S. The use of avidin-biotin interaction in immunoenzymatic techniques. J Histochem Cytochem. 1979 Aug;27(8):1131–1139. doi: 10.1177/27.8.90074. [DOI] [PubMed] [Google Scholar]
  8. Huettinger M., Schneider W. J., Ho Y. K., Goldstein J. L., Brown M. S. Use of monoclonal anti-receptor antibodies to probe the expression of the low density lipoprotein receptor in tissues of normal and Watanabe heritable hyperlipidemic rabbits. J Clin Invest. 1984 Sep;74(3):1017–1026. doi: 10.1172/JCI111469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hui D. Y., Innerarity T. L., Mahley R. W. Defective hepatic lipoprotein receptor binding of beta-very low density lipoproteins from type III hyperlipoproteinemic patients. Importance of apolipoprotein E. J Biol Chem. 1984 Jan 25;259(2):860–869. [PubMed] [Google Scholar]
  10. Knight B. L., Soutar A. K. Degradation by cultured fibroblasts and macrophages of unmodified and 1,2-cyclohexanedione-modified low-density lipoprotein from normal and homozygous familial hypercholesterolaemic subjects. Biochem J. 1982 Jan 15;202(1):145–152. doi: 10.1042/bj2020145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kroon P. A., Thompson G. M., Chao Y. S. A comparison of the low-density-lipoprotein receptor from bovine adrenal cortex, rabbit and rat liver and adrenal glands by lipoprotein blotting. Biochem J. 1984 Oct 15;223(2):329–335. doi: 10.1042/bj2230329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. McFARLANE A. S. Efficient trace-labelling of proteins with iodine. Nature. 1958 Jul 5;182(4627):53–53. doi: 10.1038/182053a0. [DOI] [PubMed] [Google Scholar]
  15. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  16. Schneider W. J., Beisiegel U., Goldstein J. L., Brown M. S. Purification of the low density lipoprotein receptor, an acidic glycoprotein of 164,000 molecular weight. J Biol Chem. 1982 Mar 10;257(5):2664–2673. [PubMed] [Google Scholar]
  17. Towbin H., Gordon J. Immunoblotting and dot immunobinding--current status and outlook. J Immunol Methods. 1984 Sep 4;72(2):313–340. doi: 10.1016/0022-1759(84)90001-2. [DOI] [PubMed] [Google Scholar]
  18. Weisgraber K. H., Innerarity T. L., Mahley R. W. Role of lysine residues of plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts. J Biol Chem. 1978 Dec 25;253(24):9053–9062. [PubMed] [Google Scholar]
  19. Weisgraber K. H., Mahley R. W. Subfractionation of human high density lipoproteins by heparin-Sepharose affinity chromatography. J Lipid Res. 1980 Mar;21(3):316–325. [PubMed] [Google Scholar]

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