| Purification: | Unpurified cell lysate. HEK293 cells transfected with expression vectors harboring target gene were harvested and washed with PBS twice. The cell pastes were re-suspended with ice-cold PBS containing mammalian cell protease inhibitor cocktail and further lysed with freeze-thaw cycles. After clarifying with 20,000 g centrifugation at 4�C for 30 min, the lysate was aliquoted, lyophilized, and stored at -80�C immediately. Protein concentration was determined by BCA kit (Thermo Scientific, Inc.) using BSA as protein standard. The gene overexpression in lysate was confirmed by Western blotting using anti-His tag antibody and/or target-specific antibodies and the lysate derived from HEK293 cells transfected with the empty expression vector was used as a negative control. |
