Nanobrook 90plus particle size analyzer

Native size of Heterotrimer (NL-A-Gal3 + sfGFP-B + mRuby-C) was determined by size-exclusion chromatography and dynamic light scattering (ESI† S4). Generally, Heterotrimer was prepared by mixing 15 μM NL-A-Gal3, 15 μM sfGFP-B, and 15 μM mRuby-C at equal volumes to total of 400 μL 1x PBS. The protein mixture was loaded onto a Superdex™ 200 10/30 GL column (GE Healthcare) connected to an ÄKTA™ pure FPLC system. Protein eluting from the column was detected at 280 nm wavelength. Raw signal was normalized based on maximum signal intensity and then plotted. Native protein molecular weight was estimated via extrapolation from a size-exclusion chromatography calibration curve, which was prepared from protein standard markers (Bio-Rad, GE Healthcare, ThermoFisher). Prior to taking dynamic light scattering measurements, the protein mixture described above was filtered through a 0.2-micron syringe filter ESI† S4B. To ensure no significant amount of protein was lost due to aggregation, the molar concentration of Heterotrimer was measured before and after filtration. Measurements were taken on a NanoBrook 90Plus Particle Size Analyzer with BIC Particle Sizing Software (Brookhaven Instruments) in technical triplicate after ten 30 second runs. Hydrodynamic diameter ± standard deviation was normalized based on number-weighted size distribution.

The effective diameter of the gLtEc molecules were measured via Dynamic Light Scattering (DLS) using a NanoBrook 90Plus Particle Size Analyzer from Brookhaven Instruments (Holtsville, New York). Native LtEc and the cross-linked samples were diluted to ~20 mM concentrations in plastic cuvettes and three scans were run at 25°C and averaged to obtain effective diameter measurements.

Stock solutions (800 mg/mL) of macromolecular crowders Ficoll 400, Dextran 20,000, and Polyethylene Glycol 20,000 (PEG) were made in Tris buffer heated to 60°C. Prior to dilution into experiments, these solutions were incubated in an orbital thermomixer at 60°C and 1400 rpm, followed by bath sonication at 60°C for 1 h. Hydrodynamic radii of each crowder was determined by dynamic light scattering (NanoBrook 90plus Particle Size Analyzer, Brookhaven Instruments).

Dynamic light scattering experiments were performed on a NanoBrook 90Plus Particle Size Analyzer (Brookhaven Instruments) with a 35 mW red diode laser. The wavelength of irradiation was 640 nm. Membrane samples were prepared to a final concentration of 40 μM with 20 mM Tris·HCl, 100 mM NaCl buffer (pH 7.4). For each sample, three runs were taken at 25 °C with one minute per run. The average diameter (effective diameter) and the distribution width (polydispersity) were calculated using the 90Plus Particle Sizing Software.

Lipid vesicles of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) or total brain lipid extract (TBLE) were prepared by dissolving the lipid in buffer A. Lipid solutions were then subjected to 10 freeze/thaw cycles using liquid nitrogen followed by bath sonication for 30 min. The size and polydispersity of lipid vesicles were measured via dynamic light scattering (NanoBrook 90plus Particle Size Analyzer, Brookhaven Instruments) to verify the formation of large unilamellar vesicles (LUVs). The mean and standard deviation of vesicle sizes were determined by assuming a log-normal distribution of the DLS data.