Activated sepharose 4b

MDCK cells infected with mouse adapted, A/chicken/Thailand/NP172/2006 at MOI 1.0 were cultured in complete Dulbecco’s modified Eagle’s medium [DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Northumberland, UK), 2 mM L-glutamine, streptomycin (100 µg/mL) and penicillin (100 units/mL)] for 24 h before subjecting to sonication in phosphate buffered saline, pH 7.4 (PBS) on ice. The homogenate was centrifuged (12,000× g, 4 °C, 20 min) and the supernatant (cell lysate) containing the native M1 protein (nM1) was collected. CNBr-activated Sepharose™ 4B (GE Healthcare, Rapsgatan, Uppsala, Sweden) coupling with rabbit polyclonal immunoglubulin to M1 was used for native M1 purification. The bead was incubated with the cell lysate containing nM1 on ice for 2 h, washed and the protein bound to the affinity beads was eluted with 0.1 M glycine buffer, pH 3.0. The eluted fraction containing nM1 (determined by SDS-PAGE and protein staining) was neutralized by using 1 M Tris-HCl, pH 9.0.

Recombinant CEF at a concentration of 2 mg/ml in PBS (pH 8.0) was mixed with Cyanogen bromide (CNBr)-activated sepharose 4B (GE Healthcare, Chicago, U.S.) overnight at 4°C. The remaining active sites on the beads were then blocked in 1 ml 100 mM Tris, pH 8.0 at RT for 2 h. After washing the beads three times with PBS (pH 8.0), the protein-coupled sepharose beads were stored in PBS (pH 8.0) with 0.025% NaN₃ at 4°C.