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Title: A sensitive and rapid method for identification and characterization of low abundance receptors

An improved method for detection of low intensity radioligand-receptor complexes resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is described. (3H)Azidopine-labeled 1,4-dihydropyridine (DHP) receptor from skeletal muscle resolved by SDS-PAGE was transferred to nitrocellulose and cut into strips and individual slices were analyzed for radioincorporation by liquid scintillation counting. (3H)Azidopine-labeled DHP binding subunit migrated as a single entity with a mass of 170 kDa and was confirmed using conventional methods. Results were obtained within 4 h after resolution by SDS-PAGE compared to 3-40 days using conventional methods. In addition, detection of extremely low signals (less than 50 cpm/lane), otherwise overwhelmed by background noise using conventional methods, was possible due to removal of free ligand during electro-transfer to nitrocellulose. This technique offers a rapid sensitive, cost effective alternative to fluorography or other conventional gel slice analysis methods for detecting low intensity radiolabeled complexes resolved by SDS-PAGE.
Authors:
; ;  [1]
  1. (Univ. of Ottawa (Canada))
Publication Date:
OSTI Identifier:
7016215
Resource Type:
Journal Article
Resource Relation:
Journal Name: Analytical Biochemistry; (USA); Journal Volume: 185:1
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; PARASYMPATHOMIMETICS; RECEPTORS; CHEMICAL COMPOSITION; AFFINITY; ELECTROPHORESIS; LIGANDS; MUSCLES; PEPTIDE HYDROLASES; RABBITS; SCINTILLATION COUNTING; TRACER TECHNIQUES; TRITIUM COMPOUNDS; ANIMALS; AUTONOMIC NERVOUS SYSTEM AGENTS; COUNTING TECHNIQUES; DRUGS; ENZYMES; HYDROGEN COMPOUNDS; HYDROLASES; ISOTOPE APPLICATIONS; MAMMALS; MEMBRANE PROTEINS; ORGANIC COMPOUNDS; PROTEINS; VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques