Bic particle sizing software

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.