Ds 11 uv vis spectrophotometer

To determine saturation concentrations in vitro, cGAS proteins and DNA mixtures were prepared at indicated protein concentrations with the same DNA concentrations using a reaction buffer of 20 mM Tris-HCl (pH 7.5), 15 mM NaCl, 135 mM KCl, 1 mg mL⁻¹ BSA, and 1 mM TCEP. Phase separation was induced at 25°C for 30 min to fully equilibrate samples and reactions were then analyzed by measuring either turbidity or fluorescence intensity. For turbidity measurements, light scattering was quantified as the absorbance at 340 nm at 25°C using a DeNovix DS-11+ UV-Vis spectrophotometer with a 10 mm optical path length. The relative saturation concentrations were determined by a two-tailed T test of the adjacent values across each pair of points in the protein/DNA titration. For fluorescence intensity analysis, microscopy images were acquired at 25°C using a Leica TCS SP5 X (Leica Microsystems) mounted on an inverted microscope (DMI6000; Leica Microsystems) with an oil immersion 63×/numerical aperture 1.4 objective lens (HCX PL APO; Leica Microsystems). For each field of view, phase separation was quantified with FIJI (Schindelin et al., 2012 (link)) by measuring total fluorescence intensity for reach field of view. The relative saturation concentrations were determined by a two-tailed T test of the adjacent values across each pair of points in the protein/DNA titration.