Purification of STAT1α and PARP-1 expressed in Sf9 insect cells

Sf9 insect cells, cultured in SF-II 900 medium (Invitrogen, 10902096), were transfected with 1 μg of bacmid driving expression of Flag-tagged STAT1α or Flag-tagged PARP-1 using Cellfectin transfection reagent (Invitrogen, 10362100) according to the manufacturer’s protocol. After five hours, the medium was supplemented with 10% FBS, penicillin, and streptomycin, and the cells were incubated for three days. The culture medium was collected as a baculovirus stock after 72 h. After three rounds of amplification of the stock, the resulting high titer baculovirus was used to infect fresh Sf9 cells to induce protein expression. After 48 h of infection, the cells were collected by centrifugation. The cells were resuspended in Flag Lysis Buffer (20 mM HEPES pH 7.9, 0.5 M NaCl, 4 mM MgCl₂, 0.4 mM EDTA, 20% glycerol, 250 mM nicotinamide, 2 mM β-mercaptoethanol) containing 2 mM sodium fluoride, 2 mM sodium orthovanadate, and 2x protease inhibitor cocktail and then lysed by Dounce homogenization and sonication. The lysate was clarified by centrifugation at 26,800 RCF for 30 min at 4 °C in a Sorvall centrifuge, transferred to a fresh tube, and mixed with an equal volume of Flag Dilution Buffer (20 mM HEPES pH 7.9, 10% glycerol, 0.02% NP-40). The diluted lysate was mixed with anti-Flag M2 agarose resin and incubated for 3 h at 4 °C with gentle mixing.

After incubation, the resin was washed as follows: (1) twice with Flag Wash Buffer #1 (20 mM HEPES pH 7.9, 150 mM NaCl, 2 mM MgCl₂, 0.2 mM EDTA, 15% glycerol, 0.01% NP-40, 0.2 mM β-mercaptoethanol) containing with 100 mM nicotinamide, 1 mM PMSF, 1 μM aprotinin, 100 μM leupeptin, 1 mM sodium fluoride, and 1 mM sodium orthovanadate, (2) twice with Flag Wash Buffer #2 [20 mM HEPES pH 7.9, 1 M NaCl (for PARP-1) or 0.5 M NaCl (for STAT1α), 2 mM MgCl₂, 0.2 mM EDTA, 15% glycerol, 0.01% NP-40, 0.2 mM β-mercaptoethanol] containing100 mM nicotinamide, 1 mM PMSF, 1 μM aprotinin, 100 μM leupeptin, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and (3) twice with Flag Wash Buffer #3 (20 mM HEPES pH 7.9, 200 mM NaCl, 2 mM MgCl₂, 0.2 mM EDTA, 15% glycerol, 0.01% NP-40, 0.2 mM β-mercaptoethanol, 1 mM PMSF). The Flag-tagged PARP-1and STAT1α proteins were eluted from the anti-Flag M2 agarose resin with Flag Wash Buffer #3 containing 0.2 mg/mL 3x Flag peptide, flash frozen in liquid N₂, and stored at −80 °C.