Ni nta magnetic agarose

Unless otherwise noted, 2.4 μM protein was incubated for 30 minutes at room temperature with a pBR322 derivative of varying topologies in 20 mM Tris, pH 7.5, 150 mM NaCl, 1mM DTT). Topology was altered by restriction enzyme digest. Protein was precipitated using Ni-NTA magnetic agarose (Qiagen 36113), then washed 2x with either a high salt (600 mM NaCl) or a low salt buffer (150 mM NaCl). Reactions were quenched with a stop buffer (20 mM Tris pH 8, 2% SDS, 1 mM EDTA) and samples were eluted by proteinase K digestion. For the re-linearization assay, 400 nM MORC-1 was bound to 50 ng total pBR322 pre-modified with Nt.BsmAI. Protein was precipitated as described above, then washed with 20 mM Tris pH 7.5, 500 mM NaCl, 1 mM DTT, then buffer exchanged into a Restriction Enzyme (RE) buffer (New England Biosciences CutSmart Buffer, B7204) and digested for 30 minutes room temperature, then washed again with either a low salt buffer (150 mM NaCl) or a high salt buffer (500 mM NaCl.) For assays with nucleotide, all buffers were supplemented with 4 mM MgCl₂. Reactions were loaded on a 1% 1xTAE gel and run overnight at room temperature, then post stained using ethidium bromide, and imaged using a Gel Logic 212 Pro (Carestream).

Phosphopeptide enrichment was done by immobilized metal affinity chromatography (IMAC). 200 μg of peptides were resuspended in 150 μl 80% acetonitrile, 0.1% trifluoroacetic acid. To prepare IMAC slurry, Ni-NTA magnetic agarose (Qiagen) was stripped with 40 mM EDTA for 30 min, reloaded with 10 mM FeCl₃ for 30 min, washed 3 times and resuspended in 80% acetonitrile, 0.1% trifluoroacetic acid. Phosphopeptide enrichment was performed using a KingFisher Flex robot (Thermo Scientific) programmed to incubate peptides with 150 μl 5% bead slurry for 30 min, wash 3 times with 150 μl 80% acetonitrile, 0.1% trifluoroacetic acid, and elute with 60 μl 1:1 acetonitrile:1% ammonium hydroxide. The eluates were acidified with 30 μl 10% formic acid, 75% acetonitrile, dried by vacuum centrifugation, and stored at −80ᵒC until mass spectrometry analysis. Both sample processing and phosphopeptide enrichment were performed in technical duplicate.