An50ti 8 hole rotor

Experiments were performed on a Beckman Coulter Optima XL-I analytical ultracentrifuge (Beckman-Coulter, Palo Alto, CA, USA) equipped with UV-visible light absorbance and interference optics detection systems, using an An50Ti 8-hole rotor and 12-mm-path-length charcoal-filled epon double-sector centerpieces. The experiments were carried out at 10°C using 5 μM to 40 μM McpN-LBD in the absence and presence of 0.6 mM NaNO₃.
Sedimentation velocity (SV) runs were carried out at a rotor speed of 48,000 rpm using 400-µl samples with the dialysis buffer as the reference. A laser was used at a wavelength of 235 nm in the absorbance optics mode. Least-squares boundary modeling of the SV data was used to calculate sedimentation coefficient distributions with the size-distribution c(s) method (74 (link)) implemented in SEDFIT v14.1 software. Buffer density (ρ = 1.003 g/ml [0.99989 g/ml in the presence of NaNO₃]) and viscosity [η = 0.013137 poise [0.01313 poise in the presence of NaNO₃]) at 10°C were estimated using SEDNTERP software (75 ) for the buffer components. The partial specific volume used was 0.7192 ml/g as calculated from the amino acid sequence using SEDNTERP software.

Experiments were performed on a Beckman Coulter Optima XL-A analytical ultracentrifuge (Beckman-Coulter, Palo Alto, CA, USA) equipped with UV-visible absorbance detection system, using an An50Ti 8-hole rotor and 12 mm path-length charcoal-filled epon double-sector centrepieces. The experiments were carried out at a rotor speed of 48 000 rpm and 7 °C using 400 µL samples of proteins dialyzed into in PIPES buffer (20 mM PIPES, pH 7.0). Protein was at 5–20 μM and L-malic acid (stock solution made up in dialysis buffer) was added at a final concentration of 1 mM. Dialysis buffer with and without ligand were used as reference. Light at a wavelength of 234 nm was recorded in the absorbance optics mode. A least squares boundary modelling of the data was used to calculate sedimentation coefficient distributions with the size-distribution c(s) method implemented in the SEDFIT v11.71 software^{82 (link)}. The Svedberg equation allowed us to estimate the experimental molecular weight from the sedimentation and diffusion coefficients obtained. Buffer density (ρ = 1.0015 g/mL) and viscosity (η = 0.01449 Poise) at 7 °C were calculated from the buffer composition using SEDNTERP software⁸³ . This software was also used to calculate the partial specific volume (0.721 ml/g) and the molecular weight (21.5 kDa) of PA2652-LBD from its sequence.

Sedimentation velocity experiments using PMCA products were performed using a Beckman Coulter XL‐A analytical ultracentrifuge equipped with absorbance optics and an An50 Ti 8‐hole rotor using similar methods as described previously (Atkinson et al., 2012; Burgess et al., 2008; Gupta, Soares da Costa, Faou, Dogovski, & Perugini, 2018; Peverelli, Soares da Costa, Kirby, & Perugini, 2016). Briefly, 2‐channel epon centrepiece quartz cells were loaded with 300–380 μl of α‐synuclein (initial protein concentration of 14 μM) and 320–400 μl of reference buffer (2.7 mM potassium chloride, 10 mM phosphate buffer, 0.29 M sodium chloride, pH 7.3–7.5). Data (up to 200 scans) were collected at 37°C continuously in absorbance mode using a wavelength of either 230 nm or 233 nm, rotor speed of 10 000 rpm, radial range of 6.0–7.3 cm, and radial step size of 0.003 cm without averaging. Multiple time‐staggered sedimentation velocity scans were analysed with the enhanced van Holde‐Weischet (vHW) method using the software UltraScan3 v4.0 (release 5699: http://www.ultrascan.aucsolutions.com/) (Demeler & van Holde, 2004) and apparent sedimentation coefficient distributions reported.