Proteomelab xl 1

Two types of sedimentation experiments were performed using a ProteomeLab XL-1 ultracentrifuge (Beckman Instruments). Prior to each experiment, the aggregate was removed from the protein sample using a Superdex 200 Increase 10/300 GL column in 25 mM HEPES (pH 7.5), 150 mM NaCl, and 0.5 mM TCEP. The first experiment assessed sample purity using sedimentation velocity. These experiments were conducted in an An50 Ti rotor at 42000 rpm and 20°C. Samples were allowed to equilibrate at 20°C for 1 h prior to the start of the experiment. Absorbance data were collected at 280 nm and fit as a function of sedimentation coefficient distributions [c(s) analysis] in SedFIT.^{36 (link),37 (link)} SedNTerp was used to calculate the partial specific volume (0.72846 mL/g), buffer density (1.0064 g/mL), and buffer viscosity (0.010034 cP) parameters.³⁸ The second set of experiments used high-speed sedimentation equilibrium and was performed to determine the binding affinity of the BECN1 homodimer.^{39 (link)} These experiments were conducted at 4°C using an An60 Ti rotor at 9500, 12000, and 19000 rpm. SedNTerp was used to calculate the partial specific volume, buffer density, and buffer viscosity parameters used for fitting.³⁸ Absorbance data were collected at 280 nm. Data were fit using SedPHAT.^{37 (link),40 (link)} Images were generated using GUSSI.^{41 (link)}