Microscope coverslips

Mass photometry (MP) experiments were performed on a Refeyn TwoMP mass photometer (Refeyn Ltd). Microscope coverslips (24 × 50 mm, Thorlabs Inc.) were cleaned by serial rinsing with Milli-Q water and HPLC-grade isopropanol (Sigma-Aldrich) followed by drying with a filtered air stream. Silicon gaskets (Grace Bio-Labs) to hold the sample drops were cleaned in the same procedure immediately before measurement. All MP measurements were performed at room temperature using Dulbecco’s PBS (DPBS) without calcium and magnesium (ThermoFisher). The instrument was calibrated using a protein standard mixture: β-amylase (Sigma-Aldrich, 56, 112, and 224 kDa), and thyroglobulin (Sigma-Aldrich, 670 kDa). Before each measurement, 15 μl of DPBS buffer was placed in the well to find focus. The focus position was searched and locked using the default droplet-dilution autofocus function after which 5 μl of protein was added and pipetted up and down to briefly mix before movie acquisition was promptly started. Movies were acquired for 60 s (6000 frames) using AcquireMP (version 2.3.0; Refeyn Ltd) using standard settings. All movies were processed and analyzed using DiscoverMP (version 2.3.0; Refeyn Ltd).

MP experiments were performed on a Refeyn TwoMP mass photometer (Refeyn Ltd, Oxford, UK). Microscope coverslips (24 mm × 50 mm, Thorlabs Inc.) were cleaned by serial rinsing with Milli-Q water and HPLC-grade isopropanol (Sigma Aldrich) followed by drying with a filtered air stream. Silicon gaskets (Grace Bio-Labs) to hold the sample drops were cleaned in the same procedure immediately prior to measurement. All MP measurements were performed at room temperature using buffer (20 mM HEPES, pH 7.5 and 150 mM NaCl). The instrument was calibrated using a protein standard mixture: β-amylase (Sigma-Aldrich, 56, 112 and 224 kDa), and thyroglobulin (Sigma-Aldrich, 670 kDa). Before each measurement, 15 μL of buffer was placed in the well to find focus. The focus position was searched and locked using the default droplet-dilution autofocus function after which 5 μL of protein samples (40 nM) was added and pipetted up and down to briefly mix before movie acquisition was promptly started. Movies were acquired for 60 s (3000 frames) using AcquireMP (version 2.3.0; Refeyn Ltd) using standard settings. All movies were processed and analyzed using DiscoverMP (version 2.3.0; Refeyn Ltd).