Isopropyl 1 thio β d galactopyranoside iptg

Escherichia coli BL21(DE3) cells were transformed with pET-32a(+)-zbRIG-I or pET-32a(+) plasmids, respectively. Then, cells were grown in 50 ml of LB medium (Beyotime) containing 0.5 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) (Sigma) at 18°C overnight with shaking at 120 rpm. Cells were pelleted by centrifugation at 4,500 rpm for 30 min and lysed in 10 ml of lysis buffer (100 mM sodium phosphate, pH 8.0, 600 mM NaCl, and 0.02% Tween-20) (Beyotime) via sonication on ice. The sonicated mixture was centrifuged at 15,000 rpm at 4°C for 20 min, and then the supernatant was affinity-purified with Dynabead His-Tag magnetic beads (Invitrogen) according to the manufacturer's instruction.
His pull-down assays were performed as described previously with some modifications (20 (link)). His-zbRIG-I-magnetic beads were incubated with the lysates of HEK 293T cells transfected with pCMV-Flag-zbTRIM25 or pCMV-Flag empty vectors on a roller, respectively. After incubation at 4°C overnight, the magnetic beads were washed three times with lysis buffer to remove unbound His-zbRIG-I and then analyzed via Western blotting using anti-Flag or anti-His antibodies. His tag protein alone was served as a negative control.

Protein construct was expressed in BL21(DE3) E. coli cells (Novagen). Transformed BL21(DE3) cells were cultured in Luria Bertani broth at 37°C and later induced with 1mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) (Sigma) when the OD₆₀₀ was around 0.6–0.8 and incubated for 4 hours post induction at 37°C. The recombinant protein EhP3 was expressed in a soluble form in the bacterial cytosol. The total induced cell pellet was harvested by high speed centrifugation and resuspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM Imidazole, containing 0.5 mg/ml lysozyme and protease inhibitors). Protein was then purified from the supernatant by immobilized metal affinity chromatography (Ni-NTA, G-Biosciences) with 250 mM imidazole in 50 mM Tris (pH 8.0) and 300 mM NaCl. The Ni-NTA purified fractions was analyzed by SDS–PAGE and further purified to homogeneity on Q-Sepharose column (XK16/20, GE Healthcare) by anion exchange chromatography using a step gradient of NaCl (100–500 mM) and 50 mM Tris (pH 8.0) as described previously [55 (link), 56 (link)]. The purified Q-Sepharose elutes was then dialyzed and concentrated using 3 kDa molecular weight cut-off centricons (Millipore) and purity and concentration of the recombinant protein was determined by SDS-PAGE and BCA respectively.