Volume 49, Issue 3 pp. 939-943

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Immunoprecipitation of an Affinity-Alkylated Fragment of the Muscarinic Acetylcholine Receptor with an Anti-Ligand Monoclonal Antibody

Marjorie W. Gainer Norman,

Marjorie W. Gainer Norman

Department of Pharmacology, University of Washington, Seattle, Washington, U.S.A.

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Neil M. Nathanson,

Corresponding Author

Neil M. Nathanson

Department of Pharmacology, University of Washington, Seattle, Washington, U.S.A.

Address correspondence and reprint requests to Dr. N. M. Nathanson at Department of Pharmacology, SJ-30, University of Washington, Seattle, WA 98195, U.S.A.Search for more papers by this author

Marjorie W. Gainer Norman,

Marjorie W. Gainer Norman

Department of Pharmacology, University of Washington, Seattle, Washington, U.S.A.

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Neil M. Nathanson,

Corresponding Author

Neil M. Nathanson

Department of Pharmacology, University of Washington, Seattle, Washington, U.S.A.

Address correspondence and reprint requests to Dr. N. M. Nathanson at Department of Pharmacology, SJ-30, University of Washington, Seattle, WA 98195, U.S.A.Search for more papers by this author

First published: September 1987

https://doi.org/10.1111/j.1471-4159.1987.tb00984.x

Citations: 5

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Abstract

Abstract: A monoclonal antibody raised against the muscarinic acetylcholine affinity-alkylating antagonist propylben-zilylcholine mustard was tested for its ability to recognize affinity-alkylated muscarinic receptors. We demonstrate here that although the antibody will not recognize the mustard when it is covalently linked to the native muscarinic receptor, trypsinization of affinity-labeled membranes releases a proteolytic labeled fragment that can be specifically immunoprecipitated by the antibody. Electrophoretic analysis of the immunoprecipitate indicates that the ligand was associated with a polypeptide of molecular weight 5,000. The recognition of this fragment by the antibody provides a means to immunopurify a portion of the muscarinic receptor that is at or near the ligand binding site.

Abbreviations used:

ELISA: enzyme-linked immunosorptive assay
mAb: monoclonal antibody
mAChR: muscarinic acetylcholine receptor
PBS: phosphate-buffered saline
PMSF: phenylmeth-ylsulfonyl fluoride
PrBCM: propylbenzilylcholine mustard
SDS: sodium dodecyl sulfate

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Volume49, Issue3

September 1987

Pages 939-943

Immunoprecipitation of an Affinity-Alkylated Fragment of the Muscarinic Acetylcholine Receptor with an Anti-Ligand Monoclonal Antibody

Abstract

Abbreviations used:

References

Citing Literature

References

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Immunoprecipitation of an Affinity-Alkylated Fragment of the Muscarinic Acetylcholine Receptor with an Anti-Ligand Monoclonal Antibody

Abstract

Abbreviations used:

References

Citing Literature

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