Instantblue solution

Protein samples were mixed with an equal amount of 2× Laemmli reducing buffer and boiled for 10 min before being subjected to SDS-PAGE analysis on 12% polyacrylamide gels. Proteins were stained with InstantBlue solution (Expedeon) for visualization or were transferred onto PVDF membranes using iBlot Dry Blotting System (Thermo Fisher Scientific) at 20 V for 7 min. Following protein transfer, the PVDF membranes were immediately blocked with 1× casein blocking buffer (Sigma-Aldrich) and were probed with either horseradish peroxidase (HRP)-conjugated primary antibodies against His tag (Qiagen) or with rabbit primary antibodies against HA-tag (Sigma-Aldrich) followed by anti-rabbit HRP-conjugated secondary antibodies. Membranes were thoroughly rinsed with PBS containing 0.1% Tween 20 between the incubations. Chemiluminescent signals were detected using Amersham ImageQuant 800 System (GE Healthcare) after a brief incubation of the PVDF membranes with Immobilon Forte Western Chemiluminescent HRP Substrates (EMD Millipore). For reblotting with a different antibody after image acquisition, the membranes were incubated with Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific) for 30 min at room temperature, rinsed thoroughly before being blocked with casein blocking buffer, and probed with antibodies as described above.

A 50 µL mixture of 10 µM ParB (C297S) or (C297S Q35C) ± 1 mM NTP ± 0.5 µM parS dsDNA (22 bp) was assembled in a reaction buffer [10 mM Tris-HCl pH 7.4, 100 mM NaCl, and 1 mM MgCl₂] and incubated for 15 min (Figure 6—figure supplement 2B) or for 1, 5, 10, 15, and 30 min (Figure 6B) at room temperature. BMOE (1 mM final concentration from a 20 mM stock solution) was then added, and the reaction was quickly mixed by three pulses of vortexing. SDS-PAGE sample buffer containing 23 mM β-mercaptoethanol was then added immediately to quench the crosslinking reaction. Samples were heated to 50°C for 15 min before being loaded on 12% TruPAGE Tris-Glycine Precast gels (Sigma Aldrich). Each assay was triplicated. Gels were stained with InstantBlue solution (Expedeon) and band intensity was quantified using Image Studio Lite version 5.2 (LI-COR Biosciences). The crosslinked fractions were averaged, and their standard deviations calculated in Excel.

Isoelectric focusing was accomplished in PhastSystem (GE Healthcare) at 15°C. The pH gradient was formed on the PhastGel IEF media 3–10.5 (pH range 3–10.5) containing pharmalyte carrier ampholytes for 75 Vh at the voltage of 2000 V. Samples were applied to the gel for 15 Vh at 200 V, and they were then allowed to migrate to their isoelectric points for 410 Vh at 2000 V. After migration, the gel was fixed with fixing solution (20% TCA, Sigma Aldrich) followed by washing with 30% methanol and 10% acetic acid in distilled water. The staining was done in InstantBlue solution (Expedeon) for 1 hour followed by washing with distilled water until the desired background was achieved.