Analyzer infinite 200pro

Fluorescence measurements were performed using a Tecan analyzer infinite 200Pro (Tecan Group Ltd, Männedorf, Switzerland). For mCherry, the excitation wavelength was 587 nm and the emission wavelength was 610 nm. For sfGFP and GFPmut3.1, the excitation wavelength was 485 nm and the emission wavelength was 520 nm. For quantification, calibration was performed using in‐house purified target proteins. We purified mCherry with immobilized metal affinity chromatography using His‐Tag‐specific binding on an Ni‐Sepharose HP column (GE Healthcare Bio‐Sciences, Uppsala, Sweden). GFP and sfGFP were purified through anion exchange chromatography using a Capto Q column (GE Healthcare Bio‐Sciences, Uppsala, Sweden), followed by hydrophobic interaction chromatography using a Butyl Sepharose HP column (GE Healthcare Bio‐Sciences, Uppsala, Sweden), and finally size exclusion chromatography using a Superdex 75 column (GE Healthcare Bio‐Sciences, Uppsala, Sweden). We applied the Beer–Lambert law to determine the concentrations of the standards using the absorbance at 280 nm and the corresponding excitation coefficients.

We performed DNA quantification using a DNA Quantitation Kit (Bio Rad, Hercules, CA), with Hoechst 33258 as dye. Depending on the expected concentration, we added 2–5 μl of sample to 200 μl fluorochrome solution (2 μg dye per ml). The calibration curve was generated using Calf Thymus DNA standard (Sigma–Aldrich). We measured fluorescence (excitation at 346 nm and emission at 460 nm) using a Tecan analyzer infinite 200Pro (Tecan Group Ltd, Männedorf, Switzerland).