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 /sup 125/I-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.
- Research Organization:
- National Cancer Institute, Bethesda, MD
- OSTI ID:
- 7025439
- Journal Information:
- Anal. Biochem.; (United States), Vol. 2
- Country of Publication:
- United States
- Language:
- English
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550201* - Biochemistry- Tracer Techniques