A binding assay for the solubilized receptors of type beta transforming growth factor: adsorption and removal of free ligand by dextran-coated charcoal.
A binding assay was developed for the measurement of solubilized receptors for transforming growth factor type beta (TGF-beta). Solubilized receptors were incubated with 125I-TGF-beta, then the unbound ligand was removed by adsorption to dextran-coated charcoal. The binding of TGF-beta to solubilized receptors was saturable and specific, and increased in a linear manner with respect to the amount of membrane protein present. Crosslinking of radioactive complexes after adsorptive removal of unbound TGF-beta yielded complexes similar to affinity-labeled TGF-beta receptors from whole cells. Treatment of a 20% charcoal suspension with 0.2-0.4% dextran was optimal for the protection of receptors from adsorption to charcoal while allowing free TGF-beta to be removed; Mr approximately 250,000 dextran was most effective. This method can assay receptors from purified membranes and crude extracts of cells and tissues, and was used to demonstrate that TGF-beta receptors are glycosylated and retain a high affinity (Kd approximately 530 pM) for ligand after solubilization.
D000327:Adsorption; D000818:Animals; D002478:Cells, Cultured; D002606:Charcoal; D003432:Cross-Linking Reagents; D003911:Dextrans; D007457:Iodine Radioisotopes; D037102:Lectins; D008024:Ligands; D051379:Mice; D010455:Peptides / Q000032:analysis*; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D018125:Receptors, Transforming Growth Factor beta; D015291:Transforming Growth Factors
D003432:Cross-Linking Reagents; D003911:Dextrans; D007457:Iodine Radioisotopes; D037102:Lectins; D008024:Ligands; D010455:Peptides; D011956:Receptors, Cell Surface; D018125:Receptors, Transforming Growth Factor beta; D002606:Charcoal; D015291:Transforming Growth Factors