Ni nta magnetic agarose beads

Recombinant VDAC1 was immobilized onto Ni-NTA Magnetic Agarose Beads (Qiagen) by exploiting its 6xHis-tag, according with manufacture’s protocol. For each experiment, a large excess of VDAC1 (30 μM) were incubated with 50 μL of beads suspension in Interaction Buffer (IB) (300 mM NaCl, 50 mM NaH₂PO₄, 20 mM imidazole, pH 8.0) to saturate all sites. Beads were collected using the magnet-based system DynaMag-2 (Life Technologies) and washed twice in Wash Buffer (300 mM NaCl, 50 mM NaH₂PO₄, 20 mM imidazole, 0.05% Tween-20, pH 6.3) to remove any excess of VDAC1. Beads-immobilized VDAC1 were incubated with 6.25 μM SOD1 WT or G93A, for 1 h at RT, under constant shaking, in 100 μL of IB. The assay was also performed by pre-incubating immobilized-VDAC1 for an extra 1 h with increasing concentration of purified human HK1 (0, 100, 200 μM) or NHK1 peptide (0, 5, 10, 25 μM), before SOD1 G93A addition. Beads were recovered using Dyna-Mag2 system and VDAC1-complexes were eluted from beads using 50 μL of Elution Buffer (300 mM NaCl, 50 mM NaH₂PO₄, 300 mM imidazole, pH 3.5). Any protein in VDAC1-bound and -unbound fractions was evaluated by western blot. Three independent experiments were performed for each condition tested.

In vitro protein synthesis of His6-tagged WISP1, GST and GST-Siah-1 fusion protein was performed using Expressway cell-free E. coli expression system (Invitrogen, Waltham, CA, United States), followed by purification of His6-tagged WISP1 using Ni-NTA magnetic agarose beads (Qiagen, München, Germany) and of GST and GST-Siah-1 using glutathione-Sepharose 4FF (GE Healthcare, Piscataway, NJ, United States). The eluted proteins were analyzed by immunoblot as previously described.

Fe³⁺-NTA-agarose beads were freshly prepared using Ni–NTA magnetic agarose beads (QIAGEN) for phosphopeptide enrichment. For each of the six fractions from the same TMT-6 plex, peptides were reconstituted in 135 μL IMAC binding/wash buffer (80% acetonitrile, 0.1% TFA) and incubated with end-over-end rotation with 35 μL of the 50% bead suspension for 30 min at RT. After incubation, the beads were washed four times each with 150 μL of wash buffer. Phosphopeptides were eluted from the beads using 50 μL of elution buffer (1:1 acetonitrile: 5% ammonia water in 5 mM pH 8 phosphate buffer, pH ~ 10), and acidified immediately to pH 3.5–4 with 10% TFA. Samples were dried using a Speed-Vac and later reconstituted with 20 μL of 3% acetonitrile, 0.1% formic acid for LC–MS/MS analysis.

The Sso strains PH1-16 [pMJ05-SmAP1-His] and PH1-16 [pMJ05-SmAP2-His] were grown in Brock’s medium (composed of Brock’s salts and supplemented with 0.2% NZamine and 0.2% sucrose) until the cells reached an OD₆₀₀ of 0.8. The cells were pelleted, washed twice with water and transferred to 400 mL Brock’s medium supplemented with 0.2% Nzamine and 0.2% D-arabinose (OD₆₀₀ = 0.2). When the cells reached an OD₆₀₀ of 0.8, the cells were harvested and lysed by sonication in 20 mL lysis buffer (50 mM Tris-HCl, pH 8.0; 0.3 M NaCl; 15 mM imidazole; 10 mM β-mercaptoethanol; 1mM PMSF). The lysate was centrifuged at 25,000 g and the supernatant was incubated with 200 µL NiNTA magnetic agarose beads (Qiagen, Hilden, Germany) overnight at 4 °C. Then, the beads were pelleted using a magnetic device. To remove non-specifically bound or weakly associated proteins or RNA, the beads were washed three times with 1 mL buffer (50 mM Tris-HCl, pH 8.0; 1 M NaCl; 40 mM imidazole in DEPC-water). RNA and protein(s) were eluted with 500 µL elution buffer (50 mM Tris-HCl, pH 8.0; 300 mM NaCl; 250 mM imidazole in DEPC-water). Fifteen microliters of the eluates were separated by SDS-PAGE (Figure 4A), whereas the rest of the eluates was extracted twice with phenol/chloroform and precipitated. The precipitated RNA was resuspended in DEPC-water.

Recombinant hexahistidine–tagged p32 was bacterially expressed and purified as previously described [23 (link)]. For protein binding assays, Ni-NTA magnetic agarose beads (Qiagen, Germany) in binding buffer (50 mM Tris pH 7.4, 150 mM NaCl, 5mM imidazole) were coated with p32 protein at 15 μg of protein/10μL beads. Radiolabeled polymersomes were incubated with the p32-coated beads in binding buffer containing 1% BSA at room temperature for 1 h. The magnetic beads were washed with binding buffer and resuspended in a final volume of 1mL of binding buffer. The radioactivity of each sample was quantified by automatic gamma counter (2470 Wizard 2, Perkin Elmer).

For in vitro histone acetyltransferase activity assay, vectors of HAG704, ACLA2, ACLB-3×FLAG, and H4-6×His were transformed into tobacco (Nicotiana benthamiana) leaf epidermal cells. 48 h after the transfection, the transfected tobacco leaves were collected and ground into powder by liquid nitrogen. Then the proteins were extracted with lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet™ P40 Substitute) and purified with Ni-NTA Magnetic Agarose beads (QIAGEN, 36113). The extracted proteins were boiled at 90 °C for 10 min and then analyzed using Western blotting. The primary antibodies used were anti-HAG704 (1:1000, in house), anti-H3 (1:1000, Abcam, ab1791), anti-H3K9ac (1:1000, Millipore, 07–352), anti-H3Kac (1:1000, Millipore, 17-615), anti-H4 (1:1000, Abcam, ab177840), anti-H4K12ac (1:1000, PTM-Biolab, PTM-121), anti-H4K5ac (1:1000, Millipore, 07-327), anti-H4K16ac (1:1000, Millipore, 07-329), anti-ACLA2 (1:1000, homemade), anti-FLAG (1:1000, Sigma, F3165). The secondary antibodies used were peroxidase-conjugated goat anti-rabbit antibody (1:10000, Abbkine, A21020), and peroxidase-conjugated goat anti-mouse antibody (1:10000, Abbkine, A21010). The original raw blotting images are shown as a Source Data file.