Discovermp

Mass photometry measurements were performed on a 2MP-0132 mass photometer (Refeyn Ltd). For the measurements, coverslips (No. 1.5 H thickness, 24 × 50 mm, VWR) were cleaned by dipping it into iso-propanol and Milli-Q water followed by drying under a stream of gaseous nitrogen. Subsequently, silicone gaskets (CultureWellTM Reusable Gasket, Grace Bio-Labs) were placed on the cleaned coverslips to create wells for samples. For mass measurements, gaskets were filled with 18 μl protein elution buffer to allow focusing the microscope onto the coverslip surface. Subsequently, 2 μl (50 nM) protein solution was added into the 18 μl droplets and mixed. A movie was recorded for 1 min using the software AcquireMP (Refeyn Ltd). Data analysis was performed using DiscoverMP (Refeyn Ltd). To convert the measured optical reflection-interference contrast into a molecular mass, a known protein size marker (NativeMarkTM Unstained Protein Standard, Invitrogen) was used.

Mass photometry data was collected on a Refeyn One^MP instrument using ReFeyn Acquire^MP software. The instrument was calibrated with a protein standard (made in-house). The following masses were used to generate a standard calibration curve: 73, 149, 479, and 800 kDa. Borosilicate coverslips were extensively cleaned with Milli-Q water and isopropanol prior to the measurements. sMAC (5 µl) was applied to 10 µl buffer (10 mM sodium phosphate, 145 mM NaCl, pH 7.3) on a coverslip resulting in a final concentration 13 µg/ml. Movies were acquired by using ReFeyn Acquire^MP software for 6000 s with a frame rate of 100 Hz. The particle landing events detected were 5480 (5241 binding and 239 unbinding). All data was processed in ReFeyn Discover^MP software. Masses of sMAC complexes were estimated by fitting a Kernel density distribution to the landing events. Gaussian fit to the mass of GroEL from the protein standard (800 kDa) was generated for peak width reference.

The MP experiments were conducted on Refyn Two-MP instrument (Refyn Ltd., Oxford, UK) on pre-cleaned coverslips (24 mm × 50 mm, Thorlabs Inc., Newton, NJ, USA) with serial washing with deionized water and isopropanol followed by drying. The silicon gaskets (Grace Bio-Labs, Bend, OR, USA) were cleaned in a similar process as coverslips and were placed onto coverslips for the experiments. The MP measurements were performed in an MP buffer containing 20 mM Tris pH 7.4, 100 mM KCl, 1 mM EDTA, and 10 mM MgCl₂. The calibration was performed using a protein standard mixture: of β-amylase (Sigma-Aldrich, 56, 112, and 224 kDa, St. Louis, MO, USA), and thyroglobulin (Sigma-Aldrich, 670 kDa). Before each experiment, 15 μL buffer was placed into a chamber formed by coverslip-Gasket and focus was searched and followed by locking it using autofocus function. G4 DNA, G4P proteins or their mixtures were added to the chamber and mixed by pipetting. The movies were recorded for 60 s (6000 frames) using AcquireMP (Version 2.3.0; Refeyn Ltd., Oxford, UK) using standard settings. All movies were processed and analyzed using DiscoverMP (Version 2.3.0; Refeyn Ltd., Oxford, UK). Individual molecular weights readings for each experiment were binned into 3 kDa intervals, plotted as histograms and fitted to multiple Gaussians using GraphPad Prism.

The measurements were performed on glass coverslips and recorded on a mass photometer (MP_TWO; Refeyn Ltd) (32 (link)) for 60 to 120 s. Each measurement was repeated at least twice. The α_Mβ₂ HP and iC3b were diluted immediately prior to measurements in 20 mM Hepes (pH 7.5), 150 mM NaCl, 5 mM MgCl₂, and 1 mM CaCl₂ to a concentration of 10 nM. For 1:1 complex formation, α_Mβ₂ HP and iC3b were mixed in a 1:1 M ratio, whereas α_Mβ₂ HP and hCD11bNb1 were mixed in a 1:10 M ratio. The influence of hCD11bNb1 on α_Mβ₂ HP–iC3b complex formation was measured using molar ratios of hCD11bNb1 to α_Mβ₂ HP (0.49, 0.74, 1.1, 1.6, 2.5, 5, and 10). The recorded videos were analyzed using DiscoverMP (Refeyn Ltd; version 2.5.0) to quantify protein-binding events. The molecular weight was obtained by contrast comparison with known mass standard calibrants measured on the same day.

Mass Photometry experiments were performed on a Refeyn TwoMP (Refeyn Ltd) (49 (link)). No.1.5, 24 mm × 50 mm microscope coverslips (Thorlabs Inc) were cleaned by serial rinsing with Milli-Q water and HPLC-grade isopropanol (Sigma Aldrich), on which a CultureWell gasket (Grace Bio-labs) was then placed. All measurements were performed at 25 °C in Dulbecco's phosphate-buffered saline without calcium and magnesium (Thermo Fisher). For each measurement, 15 μl of Dulbecco's phosphate-buffered saline buffer was placed in the well for focusing, after which 3 to 5 μl of 100 nM protein was introduced and mixed. Movies were recorded for 60 s at 50 fps under standard settings. Mass Photometry measurements were calibrated using protein standard mixture: β-Amylase (56, 112 and 224 kDa) and Thyroglobulin (670 kDa). Mass Photometry data were processed using DiscoverMP (Refeyn Ltd).
Generation of Pde6a^C857S/C857S mice is described in Supporting information text and Fig. S5. All experimental procedures involving the use of mice were performed in accordance with the National Institutes of Health guidelines and the protocol approved by the University of Iowa Animal Care and Use Committee.