Waters 626 lc system

Analytical gel filtration experiments of recombinant SIV_mac239 Nef, Hck_SH3-E, and CD3 ζ fragments were performed with a multicomponent Waters 626 LC system (Waters, MA) connected to a Superdex S75 (10/300 GL) column (GE Healthcare). Typically, 100 µl of a 1.5 mg ml⁻¹ protein solution was injected onto the column that was previously equilibrated with 10 mM Tris/HCl (pH 9.0), 100 mM NaCl buffer. Analytical gel filtration runs were done at a flow rate of 0.5 ml per minute at 293 K. The optical density was monitored at a wavelength of 280 nm. Gel filtration experiments were performed repeated times.

Analytical gel filtrations of FMNL1, FMNL2 and Cdc42 proteins were performed using a multicomponent Waters 626 LC system (Waters, MA) equipped with a Superdex S200 (10/300 GL) column (GE Healthcare). Typically, 100 μg of each protein were loaded onto the column that was equilibrated in 20 mM Tris/HCl (pH 8.0), 150 mM NaCl, 1 mM DTE buffer (supplemented with 2.5 mM MgCl₂ in the presence of Rho GTPases) prior to injection of the protein samples. Gel filtrations were run at a flow rate of 0.5 ml min⁻¹ at 293 K. The optical density was monitored at a wavelength of 280 nm over the time course of the experiment. Gel filtration experiments were performed repeatedly.