A novel method for the detection of receptors and membrane proteins by scintillation proximity radioassay.
A rapid and convenient binding assay for receptors and membrane proteins has been developed. It is based on the binding of 125I-labeled ligands to membrane proteins adsorbed to polyvinyltoluene plastic scintillation microspheres. Membranes or isolated membrane proteins adsorb to the beads upon mixing, and addition of 125I-labeled ligand induces photon emission which is proportional to the amount of added receptor or membrane protein. The interaction of acetylcholine receptor with 125I-labeled alpha-bungarotoxin and antigens with 125I-labeled antibodies or protein A were used as models to test the system. As little as 1 ng of acetylcholine receptor is detected by the assay and a linear relationship with receptor concentration is observed up to 50 ng of receptor per 250 microliter reaction medium. The effects of detergents, salts, soluble proteins, and neutral membranes were studied. Inclusion of bovine serum albumin up to 1 mg/ml, sodium chloride up to 0.5 M, and membranes up to 10 micrograms/ml cause little or no effect on the assay. Detergents at 10-fold below their critical micelle concentrations had little or no effect on the assay. The pharmacological effects of agonists such as acetylcholine were conveniently studied by following the displacement of the 125I-labeled ligand. Similarly, the amount of toxin in crude snake venom can be assayed by measuring competition with the labeled toxin. Only a few seconds are required to perform each binding assay.
Department of Biochemistry, Roche Institute of Molecular Biology, Nutley, New Jersey 07110.
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Nelson
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D007700:Kinetics; D008565:Membrane Proteins / Q000032:analysis*; D011869:Radioligand Assay / Q000379:methods; D011950:Receptors, Cholinergic / Q000032:analysis* / Q000378:metabolism; D014101:Torpedo
D002038:Bungarotoxins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D011950:Receptors, Cholinergic