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Airfuge centrifugation procedure for the measurement of ligand binding to membrane-associated and detergent-solubilized plasma membrane receptors

Abstract

A method is described in which high-speed centrifugation of membranes through an oil phase is used to separate membrane-bound and detergent-solubilized polypeptide receptor-iodinated ligand complexes from unbound ligands. Three centrifuges, the Brinkmann Eppendorf (5412), the Beckman Microfuge B and the Beckman Airfuge were evaluated for this capability. Under the conditions described, the Beckman Airfuge surpassed the others in recovering previously /sup 125/I- and /sup 32/P-labelled cell membranes. The Airfuge method was compared with the more classically employed membrane filtration method to measure specific (/sup 125/I)insulin and (/sup 125/I)thrombin binding to human placental membranes and an enriched plasma membrane fraction from mouse embryo fibroblasts, respectively, and found to be 4 to 5 times more sensitive. For example, specific binding of ligand to its receptor was demonstrated with 5 ..mu..g of protein. With slight modifications, the polyethyleneglycol 6000 method of precipitating /sup 125/I-labelled ligand-soluble receptor complexes can be adapted to the Airfuge sedimentation through oil procedure.
Authors:
Li, E L.F.; Perdue, J F [1] 
  1. Lady Davis Institute, Sir Mortimer B. Davis Jewish General Hospital, Montreal, Quebec, Canada
Publication Date:
Oct 01, 1980
Product Type:
Journal Article
Reference Number:
AIX-12-575003; EDB-81-011139
Resource Relation:
Journal Name: J. Biochem. Biophys. Methods; (Netherlands); Journal Volume: 3:4
Subject:
37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; LIGANDS; GAS CENTRIFUGATION; POLYPEPTIDES; CELL MEMBRANES; INSULIN; IODINE 125; LABELLED COMPOUNDS; PHOSPHORUS 32; RECEPTORS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; CELL CONSTITUENTS; CENTRIFUGATION; DAYS LIVING RADIOISOTOPES; ELECTRON CAPTURE RADIOISOTOPES; HORMONES; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE SEPARATION; ISOTOPES; LIGHT NUCLEI; MEMBRANES; NUCLEI; ODD-EVEN NUCLEI; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; PEPTIDE HORMONES; PEPTIDES; PHOSPHORUS ISOTOPES; PROTEINS; RADIOISOTOPES; SEPARATION PROCESSES; 400301* - Organic Chemistry- Chemical & Physicochemical Properties- (-1987)
OSTI ID:
7037329
Country of Origin:
Netherlands
Language:
English
Other Identifying Numbers:
Journal ID: CODEN: JBBMD
Submitting Site:
INIS
Size:
Pages: 207-217
Announcement Date:
Jan 01, 1981

Citation Formats

Li, E L.F., and Perdue, J F. Airfuge centrifugation procedure for the measurement of ligand binding to membrane-associated and detergent-solubilized plasma membrane receptors. Netherlands: N. p., 1980. Web. doi:10.1016/0165-022X(80)90059-7.
Li, E L.F., & Perdue, J F. Airfuge centrifugation procedure for the measurement of ligand binding to membrane-associated and detergent-solubilized plasma membrane receptors. Netherlands. doi:10.1016/0165-022X(80)90059-7.
Li, E L.F., and Perdue, J F. 1980. "Airfuge centrifugation procedure for the measurement of ligand binding to membrane-associated and detergent-solubilized plasma membrane receptors." Netherlands. doi:10.1016/0165-022X(80)90059-7. https://www.osti.gov/servlets/purl/10.1016/0165-022X(80)90059-7.
@misc{etde_7037329,
title = {Airfuge centrifugation procedure for the measurement of ligand binding to membrane-associated and detergent-solubilized plasma membrane receptors}
author = {Li, E L.F., and Perdue, J F}
abstractNote = {A method is described in which high-speed centrifugation of membranes through an oil phase is used to separate membrane-bound and detergent-solubilized polypeptide receptor-iodinated ligand complexes from unbound ligands. Three centrifuges, the Brinkmann Eppendorf (5412), the Beckman Microfuge B and the Beckman Airfuge were evaluated for this capability. Under the conditions described, the Beckman Airfuge surpassed the others in recovering previously /sup 125/I- and /sup 32/P-labelled cell membranes. The Airfuge method was compared with the more classically employed membrane filtration method to measure specific (/sup 125/I)insulin and (/sup 125/I)thrombin binding to human placental membranes and an enriched plasma membrane fraction from mouse embryo fibroblasts, respectively, and found to be 4 to 5 times more sensitive. For example, specific binding of ligand to its receptor was demonstrated with 5 ..mu..g of protein. With slight modifications, the polyethyleneglycol 6000 method of precipitating /sup 125/I-labelled ligand-soluble receptor complexes can be adapted to the Airfuge sedimentation through oil procedure.}
doi = {10.1016/0165-022X(80)90059-7}
journal = {J. Biochem. Biophys. Methods; (Netherlands)}
volume = {3:4}
journal type = {AC}
place = {Netherlands}
year = {1980}
month = {Oct}
}