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 (RayBiotech Cat#: 68QT-BCAPro-S1000
) 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.
Upon arrival, the lyophilized protein may be stored for 2 weeks at 4°C. For long term storage, it is recommended to store desiccated below -20 °C in a manual defrost freezer. Following reconstitution, the protein may be stored for 2 weeks under sterile conditions at -20 °C. For long term storage, it is recommended to make appropriate aliquots and store at -80 °C. Avoid repeated freeze-thaw cycles.