Amicon ultracel 3k centrifugal filter unit

HEK293 cells were cultured to approximately 50% confluency and transfected for 24 h with a plasmid encoding C-terminal CREB3L2 followed by a hexa-histidine tag. Cells were lysed in cold RIPA buffer supplemented with protease and phosphatase inhibitors (Pierce). The lysate was collected on ice and passed through a 21G1 needle followed by a 25G5/8 needle three to five times to shear DNA. The lysates were centrifuged at 4°C for 20 minutes at 12,000 rpm and the supernatant containing C-terminal CREB3L2 was collected. The C terminus was purified using HisPur Ni-NTA resin (Thermo) according to the manufacturer instructions for purification of His-tagged proteins by batch method. The final elution was desalted using an Amicon Ultracel-3K centrifugal filter unit (Millipore) and the purified product was measured by BCA analysis (Pierce) and confirmed by Western.

SEC was performed using an AKTApurifier FPLC System according to the manufacturer’s instructions (GE). Cells were washed twice with PBS and resuspended in hypotonic buffer (20 mM Hepes- KOH, 10 mM KCl, 1 mM MgCl₂, 1mM EDTA, 1 mM EGTA, 1 mM DTT, pH 7.5) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (PhosSTOP, Roche). Resuspended cells were subjected to three rounds of freeze thawing in liquid nitrogen. Debris was removed by centrifugation at 10,000×g for 20 min at 4 °C, followed by filtration at 0.2 μm. The column was equilibrated with gel filtration buffer (150 mM NaCl, 20 mM Hepes- KOH, 10 mM KCl, 1 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, pH 7.5). Whole cell lysates (5 mg) were applied to a S400 (HiPrep 16/60 Sephacryl) gel filtration column (Amersham Biosciences). Samples were eluted at 1 ml/min collected at every 0.75ml and monitored with an online detector at 280 nm. Every fifth sample was then concentrated using Amicon^® Ultracel^® 3K centrifugal filter units (Millipore) and analyzed by western blot.