Beckman auc sample cells

Protein samples were dialyzed into AUC buffer (20 mM HEPES, 150 mM NaCl [pH 7.5] or 50 mM Citrate, 150 mM NaCl, [pH 6.5, 5.5, 4.5, or 3.0]) prior to analysis. Sedimentation velocity ultracentrifugation experiments (AUC) were carried out using a Beckman Optima XL-I analytical ultracentrifuge (Beckman Coulter, Fullerton, CA), An60-Ti rotor, and absorbance optics. Samples were loaded into Beckman AUC sample cells with 12-mm optical path two-channel centerpieces, with matched buffer in the reference sector. Absorbance was monitored at peak wavelengths determined using a Nanodrop One spectrophotometer (Thermo). The centrifugation was performed at 48,000 rpm at 20°C, and 300–400 scans were collected at 2 min intervals over 16 h. The observed sedimentation boundaries were fitted to yield a c(s) plot according to the Lamm equation using SEDFIT [55 (link)]. Buffer densities, and protein viscosities and partial specific volumes were calculated using SEDNTERP [56 ]. Molecular mass estimates were determined after determining the coefficient of friction (c(S)) and then fitting the frictional ratio, and are based on a continuous c(M) analysis in SEDFIT.

Protein samples were used postdialysis and were never frozen. Sedimentation velocity experiments were run using Beckman Optima XL-I analytical ultracentrifuge (Beckman Coulter), An60-Ti rotor, and absorbance optics. Samples at 0.5 mg/ml were loaded into Beckman AUC sample cells with 12-mm optical path two-channel centerpieces, with matched buffer in the reference sector. Full-length ERFE constructs were centrifugated at 20,000 rpm, while ΔC1Q mutants were centrifugated at 40,000 RPM. Absorbance was measured at 230 nm to maximize signal, and the experiments were performed at 20 °C. SEDFIT was used to determine a preliminary frictional ratio using continuous c(s,ff0) distribution. The most commonly occurring frictional ratio was used as a starting value to solve for a precise frictional ratio using c(s) distributions, which yielded predicted molecular weights. In rare cases where solved c(s) and c(s,ff0) frictional ratios diverged significantly, the three most frequent c(s,ff0) frictional ratio were averaged, which ameliorated the issue. All datasets were normalized on a scale from 0 to 1, using the highest and lowest value.