TTNPB (GMP)

Labeled and unlabeled retinoids are added to nucleosol or cytosolic fractions in ethanol so that the total amount of ethanol added is constant in all tubes and did not exceed 2% of the incubation volume. The receptor preparations are incubated with retinoids at 47°C for 4-6 hr. Sephadex PD-10 desalting columns are used to separate bound radioligand from free radioligand after equilibrium is achieved. For competitive binding assays, varying concentrations of unlabeled competing ligand are incubated with the appropriate nucleosol or cytosol in the presence of a fixed concentration of [³H]tRA (sp act. 49.3 Ci/mmol) or [³H]9-cis RA (sp. act. 24.0 Ci/mmol). Final concentrations of [³H] tRA and [³H]9-cis RA for nuclear receptor binding assays are 5nM. Final concentrations of [³H]tRA for CRABP binding assays is 30 nM. The IC₅₀s are calculated. For saturation kinetics, increasing concentrations of radiolabeled ligand ([³H]tRA sp. act. 49.3 Ci/mmol, [³H]TTNPB sp. act. 5.5 Ci/mmol) are added to the nucleosol of the appropriate receptor subtype in the presence (nonspecific binding) or absence (total binding) of a 100-fold molar excess of the corresponding unlabeled retinoid. Specific binding is defined as the total binding minus nonspecific binding. Saturation kinetics are calculated^[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.