Mustang e membrane

The bacterial expression and purification of human PARP1 were carried out by following a previously published protocol.^{9 (link)} The purified protein was further passed through an acrodisc unit with mustang E membrane (Pall Corporation, Port Washington, NY) by following the manufacturer’s instructions. The final endotoxin levels (< 0.5 EU mg⁻¹ mL⁻¹) were measured using Pierce LAL chromogenic endotoxin quantitation kits (Thermo Fisher Scientific). Purified PARP1 was analyzed by SDS-PAGE, flash frozen using liquid nitrogen, and stored at −80°C.
Recombinant human GCSF was expressed through transient transfection into Expi293F cells using the polyethylenimine max (PEI-MAX) transfection regent. Culture media of Expi293F cells transfected with the expression construct were collected at days 3 and 6 post-transfection and centrifuged at 4,000×g for 30 min. The supernatant was dialyzed in PBS buffer for overnight and another 6 hours in PBS at 4 °C and then loaded on a gravity flow column packed with 2 mL of Ni-NTA agarose resin (Thermo Fisher Scientific), followed by washing with PBS containing 20 mM imidazole and eluting with PBS containing 400 mM imidazole. GCSF protein was then dialyzed in PBS buffer for overnight and another 6 hours in PBS at 4 °C and concentrated using a 10 kDa-cutoff amicon centrifugal concentrator. Purified human GCSF was analyzed by SDS-PAGE and stored at −80°C.