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.
Sunahara
R K
RK
Department of Pharmacology, University of Ottawa, Canada.
Murphy
B J
BJ
Tuana
B S
BS
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D015220:Calcium Channels; D004095:Dihydropyridines; D004591:Electrophoresis, Polyacrylamide Gel; D008722:Methods; D009132:Muscles / Q000032:analysis*; D010447:Peptide Hydrolases; D010449:Peptide Mapping; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000032:analysis*; D012588:Scintillation Counting
D000345:Affinity Labels; D001386:Azides; D015220:Calcium Channels; D004095:Dihydropyridines; D011978:Receptors, Nicotinic; C041130:azidopine; D010447:Peptide Hydrolases