Amvn viscosimeter

Sedimentation velocity experiments were performed on an Optima XL-I analytical ultracentrifuge (Beckman) using an An 60 Ti rotor and double-sector epon center pieces. The proteins were added to a 20 mM Tris + 100 mM NaCl buffer at 0.6 and 1.6 mg/ml for rHS-MMP2 and rNUCB1-His, respectively. Buffer density and viscosity were measured using a DMA 5000 densitometer and a AMVn viscosimeter, respectively (both Anton Paar). Fluorescently labeled protein concentration distribution was monitored at 544 nm at 50,000 rpm and 20°C. Time-derivative analysis was computed using the SEDFIT software package, v12.1b (Schuck, 2000 (link)), resulting in a c(s) distribution and an estimate of the molecular weight Mf (from the sedimentation coefficient and the diffusion coefficient, as inferred from the broadening of the sedimentation boundary, assuming all observed species share the same frictional coefficient f/f₀).

Sedimentation velocity experiments were performed on an Optima XL‐I analytical ultracentrifuge (Beckman Inc., Palo Alto, Ca, USA) using a 60 Ti rotor and double‐sector epon centerpieces. The CrIFT57/38 complex (0.56 mg/ml) was in a 10 mM HEPES (pH 7.5) buffer containing 100 mM NaCl, 10% glycerol and 2 mM TCEP. Buffer density and viscosity were measured using a DMA 5000 densitometer and an AMVn viscosimeter, respectively (both Anton Paar, Graz, Austria). Protein concentration distribution was monitored at 280 nm, at 235,000 g and 20°C. Time‐derivative analysis was computed using the SEDFIT software package, version 12.1b (Schuck, 2000), resulting in a c(s) distribution and an estimate for the molecular weight Mf (from the sedimentation coefficient and the diffusion coefficient, as inferred from the broadening of the sedimentation boundary, assuming all observed species share the same frictional coefficient f/f0).