Hrp conjugated anti polyhistidine antibody

Overnight cultures of E. coli BL21 Origami (DE3) containing pET46-Ek/LIC plasmids (Supplementary Table 1) were diluted 1:100 and grown to an OD_600nm of 0.5 in LB supplemented with 100 μg/ml ampicillin and 1% (w/v) glucose, at 37 °C and 200 rpm. Before induction, glucose from the media was removed and replaced by LB with 100 μg/ml ampicillin. Isopropyl β-D-thiogalactopyranoside (IPTG) was added to a final concentration of: 0.1 mM for rBapB-Spy, 1 mM for rGFP-SpyCatcher and 1 mM for rGFP. The cultures were shaken (150 rpm) at 20 °C overnight. Pellets were resuspended in BugBuster lysis buffer (Novagen) and incubated 30 min RT. Cells were sonicated (3 cycles of 30 s pot 4; 5 cycles of 30 s pot 5) and centrifuged (26,200 × g, 30 min and 4 °C). Supernatants were filtered (0.45 μm). Recombinant proteins were purified by Ni affinity chromatography using HisGraviTrap gravity-flow columns (GE Healthcare) following the manufacturer’s protocol. Detection of recombinant proteins cloned in pET46-Ek/LIC vector was assessed using mouse monoclonal HRP-conjugated anti-polyHistidine antibody (1:2000, Sigma). The concentration of purified proteins was determined using the Bradford Protein Assay (Bio-Rad) with BSA protein as a standard.

For Western blot analysis, cell nuclei were prepared by cell disruption in CSK buffer (100 mM NaCl, 300 mM Sucrose, 10 mM Tris pH 7.5, 3 mM MgCl₂, 1 mM EGTA, 1.2 mM PMSF, 0.5% Triton X-100), and centrifugation for 10 min at 4000 rpm. Nuclei were resuspended in 1× NuPAGE LDS Sample buffer (Invitrogen, NP0007) with DTT (12.5 mM), sonicated, heated for 10 min at 72°C and loaded on 4–12% gradient Novex Tris–glycine precast gels (Invitrogen, NP0315). After separation, the proteins were transferred to PVDF membranes and probed with primary antibodies against macroH2A1 at 1:1000 (Active Motif 39594) detected by an antirabbit-HRP antibody at 1:1000 (Roth 4750.1) according to the manufacturer's protocoles. Expression of the BAP-tagged proteins was detected with an HRP-conjugated anti-polyHistidine antibody at 1:4000 (Sigma A7058) and biotinylated proteins were visualized with an HRP-conjugated streptavidin at 1:1000 (Sigma S2438). For streptavidin-HRP, an additional washing step with PBS/Tween-20 0.1% and NaCl 500 mM was performed.