Goat anti mouse igg h l horseradish peroxidase hrp conjugate

Purified human α₂-macroglobulin protein (725 kDa) was obtained from Enzo Life Science (New York, USA), anti-human monoclonal mouse α₂-macroglobulin (immunoglobulin (Ig)G1 clone) from R&D Systems (Minneapolis, MN, USA), and goat anti-mouse IgG (H + L)-horseradish peroxidase (HRP) conjugate from Bio-Rad Laboratories (Hercules, CA, USA). Native PAGE™ Sample Buffer, Native PAGE Running Buffer, Dark Blue Cathode Buffer, Native Mark^™ Unstained Protein Standard, and Light Blue Cathode Buffer were obtained from Thermo Fisher Scientific (Waltham, MA, USA). DTT, Blocking One and Peroxidase Stain 3,3′-Diaminobenzidine kit (Brown Stain) were purchased from Nacalai Tesque (Kyoto, Japan). Immune-Blot^® polyvinylidene difluoride membrane, Sequi-Blot™ membrane, Precision Plus Protein™ Dual Color Standards and Mini-PROTEAN^® TGX™ were obtained from Bio-Rad Laboratories. Perfect NT Gel, Perfect NT Gel System and SDS-PAGE Running Buffer were obtained from DRC (Tokyo, Japan), and ECL Prime Western Blotting Detection Reagent from GE Healthcare (Buckinghamshire, UK).

Western blots were carried out as described previously (71 (link)) with modifications. Bacterial cultures were grown overnight in cation-adjusted Mueller-Hinton Broth (caMHB), diluted 1:100 in fresh caMHB, and grown for 16 hours to stationary phase. A total of 500 μL of the bacterial culture was pelleted (21,000 × g, 25 minutes) and resuspended in a volume of 2× Laemmli sample buffer (Bio-Rad) equal to sample OD₆₀₀/6 × 250. Samples were boiled at 100°C for 20 minutes. Fifteen microliters of each sample was loaded into the wells of a 12% polyacrylamide gel (Bio-Rad; 4561045) and run at 150 V for 75 minutes. Transfer from the gel to a nitrocellulose membrane (Bio-Rad; 1704158) was carried out using the Bio-Rad Trans-Blot Turbo transfer system. The membrane was probed with anti-RNA sigma S antibody (BioLegend; cat. number 663703) and anti-E. coli RNA Polymerase β Prime Antibody (BioLegend; cat. number 662904) as a loading control. The secondary antibody was goat anti-mouse IgG (H+L)-horseradish peroxidase (HRP) conjugate (Bio-Rad; cat. number 1706516). The blot was developed with Clarity western ECL substrate (Bio-Rad; 170-5061). Final imaging was done using the Bio-Rad ChemiDoc MP imaging system, and blot analysis was conducted using ImageJ (72 (link)).

For western blot analysis, cells were fixed with 20% trichloroacetic acid (TCA) and disrupted by bead beating. Lysate and precipitate/debris were mixed with 600 µL 10% TCA and pelleted. The pellet was resuspended in 1× Laemmli buffer with 5% β-mercaptoethanol and 160 mM Tris-HCl (pH 7.4), boiled for 10 min and sonicated briefly. The extract was directly subjected to sodium dodecyl sulfate (SDS) gel analysis without clearance. The following antibodies were used: mouse monoclonal anti-Rad53 (clone EL7, in house^{58 (link)}, 1:5), mouse monoclonal anti c-MYC (clone 9E10, Santa Cruz Biotechnology Cat# sc-40, RRID:AB_627268, 1:2000), rabbit anti-histone H3 (EpiCypher, Cat# 13-0001, 1:5000), rabbit anti-histone H4 (Abcam Cat# 7311, 1:4000), rabbit anti-histone H3 (acetyl K27) (Abcam, Cat# ab4729, 1:2000), mouse monoclonal anti-acetyl-lysine (T52, in-house^{59 (link)}, 1:10), mouse monoclonal anti-Pgk1 (novex, Cat# 459250, 1:10,000), mouse monoclonal anti-Porin (abcam, Cat# 110326, 1:1000), rabbit anti-GFP (Amsbio, Cat# TP401, 1:5000), goat anti-mouse IgG (H + L)-horseradish peroxidase (HRP) Conjugate (Bio-Rad, Cat# 1706516, 1:20,000), goat anti-rabbit IgG (H + L)-HRP Conjugate (Bio-Rad, Cat# 1706515, 1:20,000). Detection was performed by electrochemiluminescence (GE Healthcare). Uncropped western blots are shown in Supplementary Figs. 10 and 11.