Advancebio sec column

iRicKO MEFs treated with EtOH or 4-OHT were lysed in ice-cold CHAPS-containing buffer (50 mM Na-HEPES, 100 mM NaCl, 2 mM MgCl₂, 2 mM DTT, and 0.3% CHAPS, complete EDTA-free protease inhibitors). The lysate was passaged 6 times on ice through a 1 ml syringe with a 22-gauge and 27-gauge needle and cleared by ultracentrifugation at 12,000 × g for 20 min at 4^oC. The cleared lysates were normalized to a protein concentration of 4 mg/mL in the CHAPS buffer. The lysates (500 uL per sample) were filtered to remove large particles through 0.45 μm spin filters (Millipore Ultrafree-MC) at 12,000 × g for 1 min at 4^oC. Using a Thermo Dionex BioRS UHPLC system each sample kept at 4^oC was injected (350 uL per sample) onto an Agilent AdvanceBio SEC column (7.8×300 mm, 300A pores, 2.7 um particles). Prior to separations the column was cooled to 5^oC and pre-equilibrated with 10 column volumes of SEC running buffer (0.3% CHAPS, 50 mM Na-HEPES, 100 mM NaCl, 2 mM MgCl₂, 2 mM DTT). The flow rate was 0.5 mL/min and 12 fractions were collected from retention time 8.5 to 14.5 min with a total run time per sample of 45 min. The fractions in the SEC running buffer were diluted into SDS-PAGE sample buffer and equal fraction volumes loaded for western blotting.