Anti his antibody

X. laevis Nup358 constructs (residues 1–800 and 1–900) were cloned into pET21a with a C-terminal His tag. Expression was carried out in E.coli BL21 DE3. Briefly, cells were grown in terrific broth media, supplemented with 100 μg/ml of Ampicillin and 30 μg/ml of Chloramphenicol, until OD₆₀₀ reached 0.6. Cells were then transferred at 4°C for 30 min before the addition of 1 mM IPTG and incubation overnight at 18°C. Cells were pelleted at 3,000 g for 20 min and resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM TCEP, 10 mM Imidazole) supplemented with a protease inhibitor cocktail. Lysis was performed by sonication and the soluble fraction was separated by centrifugation at 40,000 g for 1 hour at 4°C. The supernatant was incubated with Ni-NTA beads pre-equilibrated with lysis buffer, and purification was performed per manufacturer’s recommendation. Eluted fractions were further separated by gel filtration chromatography with a Superdex 200 Increase 10/300 GL in gel filtration buffer (20 mM Hepes pH 7.4, 150 mM NaCl, 0.5 mM TCEP). Fractions were analyzed by Western blotting using an Anti-His antibody (Takara 631210). The Superdex 200 Increase 10/300 GL column was previously calibrated in gel filtration buffer using a high molecular weight kit from MW of 43 kDa to 669 kDa (Cytiva 28-4038-42).

Two methods were used to detect the auto-PARsylation of TNKS SAM-catalytic domains. In the first purified proteins were run on SDS-PAGE and transferred to PVDF membrane (Millipore). Ponceau staining was used to check loading of proteins. Auto-PARsylation of proteins that has occurred during expression in E. Coli was detected by Western blot with an anti-PAR polyclonal antibody (Trevigen, #4336-APC-050). In the second protocol, purified proteins, both WT and mutants, were mixed with 6-biotin-17-NAD (TREVIGEN) in the reaction buffer (50 mM Hepes pH7.5, 150 mM NaCl, 2 mM MgCl₂) at 30°C for 90 minutes. Reaction products were resolved on SDS-PAGE and detected by Western blot using HRP conjugated anti-biotin antibody (Cell Signaling Technology, #D5A7). To check loading of protein, the same membrane was stripped and probed with anti-His antibody (Takara, #631212). Histone PARsylation assay was conducted as described in HT Universal Color PARP Assay Kit w/ Histone Coated Strip Wells (Trevigen, #4677–096-K) [17 (link)].