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. 1979 Nov;40(5):701–709. doi: 10.1038/bjc.1979.250

Isolation of plasma-membrane components from cultured human pancreatic cancer cells by immuno-affinity chromatography of anti-beta 2M sepharose 6MB.

S Påhlman, I Ljungstedt-Poahlman, A Sanderson, P J Ward, A Grant, J Hermon-Taylor
PMCID: PMC2010108  PMID: 92330

Abstract

Human pancreatic exocrine adenocarcinoma cells established in tissue culture expressed both HLA and beta 2-microglobulin (beta 2M). Plasma-membrane components of this pancreatic cancer cell line were purified from plasma membrane fractions enriched by sucrose density-gradient centrifugation, using immunoaffinity chromatography on immobilized anti-human beta 2M antibody. Both rabbit and mouse monoclonal anti-beta 2M IgG were used, with a 20--25-fold overall purification of 5'-nucleotidase. The method was applicable to 5 x 10(7) cells and permitted the solubilization of membranes retained on the column, with the selective desorption of components not associated with beta 2M before the subsequent elution at pH 3 of beta 2M-associated macromolecules. The acid eluate contained one major and two minor bands in the 40--45,000 mol.-wt range with two additional enriched components of 18,000 and 22,000 dalton. A major carbohydrate-containing component of high mol. wt was also found to be associated with the pancreatic cancer-cell plasma membrane.

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Selected References

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

  1. Avruch J., Wallach D. F. Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells. Biochim Biophys Acta. 1971 Apr 13;233(2):334–347. doi: 10.1016/0005-2736(71)90331-2. [DOI] [PubMed] [Google Scholar]
  2. Axén R., Porath J., Ernback S. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature. 1967 Jun 24;214(5095):1302–1304. doi: 10.1038/2141302a0. [DOI] [PubMed] [Google Scholar]
  3. Banwo O., Versey J., Hobbs J. R. New oncofetal antigen for human pancreas. Lancet. 1974 Apr;1(7859):643–645. doi: 10.1016/s0140-6736(74)93197-3. [DOI] [PubMed] [Google Scholar]
  4. Berggård I., Bearn A. G. Isolation and properties of a low molecular weight beta-2-globulin occurring in human biological fluids. J Biol Chem. 1968 Aug 10;243(15):4095–4103. [PubMed] [Google Scholar]
  5. Beutler B., Nagai Y., Ohno S., Klein G., Shapiro I. M. The HLA-dependent expression of testis- organizing H-Y antigen by human male cells. Cell. 1978 Mar;13(3):509–513. doi: 10.1016/0092-8674(78)90324-0. [DOI] [PubMed] [Google Scholar]
  6. Brunner G., Ferber E., Resch K. Fractionation of membrane vesicles. I. A separation method for different populations of membrane vesicles of thymocytes by affinity chromatography on Con A-Sepharose. Anal Biochem. 1977 Jun;80(2):420–429. doi: 10.1016/0003-2697(77)90664-9. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Gelder F. B., Reese C. J., Moossa A. R., Hall T., Hunter R. Purification, partial characterization, and clinical evaluation of a pancreatic oncofetal antigen. Cancer Res. 1978 Feb;38(2):313–324. [PubMed] [Google Scholar]
  9. Goodfellow P. N., Jones E. A., Van Heyningen V., Solomon E., Bobrow M., Miggiano V., Bodmer W. F. The beta2-microglobulin gene is on chromosome 15 and not in the HL-A region. Nature. 1975 Mar 20;254(5497):267–269. doi: 10.1038/254267a0. [DOI] [PubMed] [Google Scholar]
  10. Grant A. G., Duke D., Hermon-Taylor J. Establishment and characterization of primary human pancreatic carcinoma in continuous cell culture and in nude mice. Br J Cancer. 1979 Feb;39(2):143–151. doi: 10.1038/bjc.1979.24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grant D. A., Magee A. I., Hermon-Taylor J. Optimisation of conditions for the affinity chromatography of human enterokinase on immobilised p-aminobenzamidine. Improvement of the preparative procedure by inclusion of negative affinity chromatography with glycylglycyl-aniline. Eur J Biochem. 1978 Jul 17;88(1):183–189. doi: 10.1111/j.1432-1033.1978.tb12436.x. [DOI] [PubMed] [Google Scholar]
  12. Hynes R. O. Alteration of cell-surface proteins by viral transformation and by proteolysis. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3170–3174. doi: 10.1073/pnas.70.11.3170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kvist S., Ostberg L., Persson H., Philipson L., Peterson P. A. Molecular association between transplantation antigens and cell surface antigen in adenovirus-transformed cell line. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5674–5678. doi: 10.1073/pnas.75.11.5674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Köhler G., Milstein C. Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur J Immunol. 1976 Jul;6(7):511–519. doi: 10.1002/eji.1830060713. [DOI] [PubMed] [Google Scholar]
  15. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  16. Ljungstedt-Påhlman I., Seiving B., Sjöholm I. Heterogeneous insulin- and concanavalin A-binding among spleen lymphocytes established by affinity chromatography. Exp Cell Res. 1977 Nov;110(1):191–200. doi: 10.1016/0014-4827(77)90285-3. [DOI] [PubMed] [Google Scholar]
  17. Neville D. M., Jr Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J Biol Chem. 1971 Oct 25;246(20):6328–6334. [PubMed] [Google Scholar]
  18. Rauch J. E., Shuster J., Thomson D. M., Gold P. Isolation of HLA and tumor antigens by means of affinity chromatography employing anti-beta2-microglobulin (beta2m) antiserum. Cancer. 1978 Sep;42(3 Suppl):1601–1605. doi: 10.1002/1097-0142(197809)42:3+<1601::aid-cncr2820420838>3.0.co;2-v. [DOI] [PubMed] [Google Scholar]
  19. Sanderson A. R. HLA "help" for human B2-microglobulin across species barriers. Nature. 1977 Sep 29;269(5627):414–417. doi: 10.1038/269414a0. [DOI] [PubMed] [Google Scholar]
  20. Tada N., Tanigaki N., Pressman D. Human cell membrane components bound to beta2-microglobulin in T cell-type cell lines. J Immunol. 1978 Feb;120(2):513–519. [PubMed] [Google Scholar]
  21. Thomson D. M., Rauch J. E., Weatherhead J. C., Friedlander P., O'Connor R., Grosser N., Shuster J., Gold P. Isolation of human tumour-specific antigens associated with beta2 microglobulin. Br J Cancer. 1978 May;37(5):753–775. doi: 10.1038/bjc.1978.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Titanji V. P., Påhlman S. Protamine-agarose non-charged alkyl derivatives of agarose in the purification of rat-liver phosphoprotein phosphatases. Biochim Biophys Acta. 1978 Aug 7;525(2):380–391. doi: 10.1016/0005-2744(78)90233-4. [DOI] [PubMed] [Google Scholar]

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