Life sciences ls55 model fluorometer

Binding of all-trans-retinol to HRASLS and HRASLS/LRAT chimeric proteins was studied by spectrofluorometry^{47 (link)}. Measurements were performed with a PerkinElmer Life Sciences LS55 model fluorometer (Watman). Interaction of all-trans-retinol with proteins was assessed by monitoring the quenching of protein fluorescence with increasing concentrations of ligand. Samples were excited at 285 nm. Emission spectra were recorded between 300 and 520 nm with bandwidths for excitation and emission set to 10 nm. All titrations were carried out at 25 °C in 20 mM Tris/HCl buffer, pH 8.0, containing 50 mM NaCl and 10% glycerol (v/v). all-trans-Retinol was delivered in methanol such that the final volume of this organic solvent did not exceed 0.5% of the sample's total volume. All binding data were corrected for background and self-absorption of excitation and emission light⁴⁸ . Apparent K_d values were calculated based on nonlinear regression of the experimental data and using one or two site saturation ligand-binding models.