Anti polyhistidine antibody

The Atg12-containing fraction eluted with 200 mM imidazole from Ni-NTA was applied to a HiTrap Q anion exchange column pre-equilibrated with 50 mM Tris, pH 8.0, and 100 mM NaCl. The column was washed with 50 mM Tris, pH 8.0, and 100 mM NaCl, and the protein was eluted using a salt gradient from 50–500 mM NaCl and then 500–1 M NaCl. Fractions containing the Atg12 protein were pooled and applied to a Superdex 75 size-exclusion chromatography column pre-equilibrated with PBS, pH 7.4, and 0.2 mM PMSF. Fractions containing Atg12 were pooled and analyzed. Proteins were detected on western blots by anti-polyhistidine antibody (Sigma-Aldrich, H1029), Atg12 antibody (Rockland/Fisher Scientific, 200-401-437), and Atg10 antisera.

The synthetic HBV ε RNA fragments shown in S1 Table were dissolved in 1×TE buffer (DEPC treated) to a concentration of 1 μg/μl and denatured in 80°C water bath for 5 min, followed by slow cooling down to room temperature for RNA annealing and secondary structures formation. HBV ε RNAs were [γ-³²P] end-labeling by T4 polynucleotide kinase (New England Biolabs) and purified by Quick Spin Sephadex G25 column (Roche).
The indicated amount of ISG20 proteins were incubated with 100 ng ³²P-radiolabeled HBV RNAs in the presence of 20 mM HEPES, 100 mM KCl, 1 mM DTT, 0.5 mg/ml BSA, 10% Glycerol and 0.05 μg/μl poly[dI-dC] at 30°C for 30 min to form nucleoprotein complexes. 1 μl of monoclonal anti-polyHistidine antibody (Clone H1029, Sigma) was used for supershifting of the His-ISG20/ HBV ε complex. 1 μg, 2 μg or 4 μg of cold unlabeled HBV ε RNAs were used to compete for binding of the ISG20 protein to 100 ng radiolabeled HBV ε. The nucleoprotein complexes were separated by native 5% PAGE at 200 V in a gel buffer containing 50 mM Tris, 45 mM Boric acid, 1% (v/v) Glycerol for 2 h in the cold room. The gel was fixed in 10% acetic acid and 10% methanol, dried, and visualized by autoradiography.

A single colony was inoculated into 5 ml of LB Agar medium containing 100 μg/mL ampicillin and incubated overnight at 37 °C and 120 rpm. Then, 250 μL of LB broth containing recombinant E. coli BL21 (DE3) was added to 25 ml of autoclaved LB medium. After the OD600 culture reached 0.6, protein expression was induced by 1 mM IPTG and incubated for 6 h at 37 °C and 120 rpm. Sampling was performed every hour from the culture. Then, samples were centrifuged at 5000 rpm for 5 min at 4 °C and the cell pellet was collected. It was sonicated at 25 W for 30 s, after resuspension in lysis buffer (20 mM NaH₂PO₄, 25 mM imidazole and 0.5 M NaCl, pH = 7.4)³² . after which the cell debris was removed by centrifugation at 4 °C at 13000 × g for 10 min, the soluble crude extract was electrophoresed by SDS-PAGE on 12% separating and 5% stacking polyacrylamide gels and Coomassie brilliant blue staining. The expression of recombinant protein was confirmed using western blot assay after transferring proteins from polyacrylamide gel onto polyvinylidene difluoride membranes using 2% BSA as a blocking solution, anti-poly-histidine antibody (Sigma, USA) and DAB colored substrate^{34 (link)}.