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Approaches to Isolating Minor Membrane Components Using Monoclonal Antibodies, as in Purifying 5′-Nucleotidase

  • J. P. Luzio
  • E. M. Bailyes
  • R. A. Daw
  • A. C. Newby
  • K. Siddle
Part of the Methodological Surveys in Biochemistry and Analysis book series (MSBA, volume 13)

Abstract

Isolation of a minor membrane protein requires optimal solubilization, then a protocol, with suitable affinity steps, that gives an active product in good purity and yield. Amphi-pathic detergents of diverse molecular structure are commonly used, aiming at “the smallest and least polydisperse active protein” [1] by criteria including non-sedimentability at high g forces and Polyacrylamide gel electrophoresis (PAGE) which also indicates the solubilized active protein’s molecular size. Immuno-affinity purification, very effective and of wide use because monoclonal antibodies can be prepared for a specific protein available only in partially purified form, entails immunization, production and purification of monoclonal antibody, preparation of immunoadsorbent, and adsorption and elution of antigen.

For 5′-nucleotidase, an intrinsic glycoprotein of the plasma membrane (p.m.) known to be an ectoenzyme in many mammalian cells, a systematically selected zwitterionic detergent can solu-bilize active enzyme from rat-liver p.m. Purification entailed an immunoaffinity step with a monoclonal antibody immunoadsorbent [2, 3]; yield 16%, enrichment ×10,000, vs. the homogenate.

There are 5 × 105 monomer molecules of 5′-nucleotidase per liver cell — of the same order as reported for insulin receptors, 1 –2.5 × 105 per liver cell. It is suggested that the systematic approach to solubilization reported and a monoclonal antibody immunoaffinity step will prove of general use in the purification of minor membrane components, e.g. insulin receptor.

Keywords

Insulin Receptor Myeloma Cell Mixed Micelle Cyanogen Bromide Intrinsic Membrane Protein 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Plenum Press, New York 1984

Authors and Affiliations

  • J. P. Luzio
    • 1
  • E. M. Bailyes
    • 1
  • R. A. Daw
    • 1
  • A. C. Newby
    • 1
  • K. Siddle
    • 1
  1. 1.Department of Clinical Biochemistry, Addenbrooke’s HospitalUniversity of CambridgeCambridgeUK

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