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Antibodies to a Covalent Antagonist used to Isolate the Muscarinic Cholinergic Receptor from Rat Brain

  • H. K. Dadi
  • R. J. Morris
  • E. C. Hulme
  • N. J. M. Birdsall
Part of the Methodological Surveys in Biochemistry and Analysis book series (MSBA, volume 13)

Abstract

To isolate cell-surface receptors a 103–106 fold purification is usually required, which necessitates the use of a powerful affinity step. For this purpose ligand affinity chromatography has proved invaluable provided that the ligand-receptor interaction is not destroyed by solubilization of the receptor or by immobilization of the ligand. The muscarinic cholinergic receptor (MCR) of brain requires over 10,000-fold purification to attain homogeneity [1] and to date a successful ligand chromatography procedure has not been devised. We have therefore pursued an alternative strategy, namely to raise antibodies to the covalent antagonist, propylbenzilylcholinemustard (PrBCM) [2], and to use these to isolate solubilized ligand-receptor complexes (PrBCM-MCR). This approach has been successful with large polypeptide ligands (e.g. insulin [3], transferrin [4], snake α-toxins for the nicotinic cholinergic receptor [5]). Its application to small ligands might be expected to encounter problems since it requires both the antibody and the receptor to bind the ligand simultaneously. However, if a covalent ligand is used the ligand-receptor complex can be denatured, exposing the ligand to the antibody without disrupting its association with the receptor.

Keywords

Sodium Dodecyl Sulphate Sodium Dodecyl Sulphate Sodium Dodecyl Sulphate Concentration Brain Membrane Fold Purification 
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

  • H. K. Dadi
    • 1
  • R. J. Morris
    • 1
  • E. C. Hulme
    • 1
  • N. J. M. Birdsall
    • 1
  1. 1.Laboratory of Neurobiology, and Division of Molecular PharmacologyNational Institute for Medical ResearchLondonUK

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