Mouse monoclonal anti acetyl lysine antibody

Loading buffer with additional β-Me (2%) to denature the protein sample, protein samples in SDS-PAGE sample buffer were boiled for 8 min, resolved on an SDS-PAGE gel, and transferred to PVDF membrane (Thermo Fisher Scientific). The membrane was blocked in 5% skim milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) at room temperature for 1 hour. Then the membrane was incubated with mouse anti-His tag antibody (1:10000, Proteintech, Cat. No. HRP-66005), mouse anti-vinculin monoclonal antibody (1:2000, Cat. No. 66305-1-Ig), mouse monoclonal anti-acetyl lysine antibody (1:1000, Cell Signaling Technology, Cat. No. 9681), mouse anti-strep-tag II monoclonal antibody (1:2000, Abbkine, Cat. No. ABT2230), rabbit polyclonal FtsZ antibody (1:1000, CUSABIO, Cat. No. CSB-PA359270HA01EGX) in TBST at 4 °C overnight. The membrane was washed with TBST (4 × 5 min) before the addition of the secondary goat anti-mouse horseradish peroxidase conjugate (1:4000, Proteintech, Cat. No. SA00001-1) or HRP-conjugated goat anti-rabbit antibody (1:5000, Cell Signaling Technology, Cat. No. 7074). After 1 hour, the membrane was washed with TBST (4 × 5 min). Final detection of HRP activity was performed using ECL Plus chemiluminescent substrate (Thermo Fisher Scientific). The image was acquired with Image Quant LAS 4000 or ChemiScope 6300. Uncropped images can be found in the Source Data File.

Purified E. coli topoisomerase I [35 (link)] was incubated at 0.1 μg/μL with 5 mM acetyl phosphate at 37 °C in previously published conditions [21 (link),36 (link)] of 150 mM Tris-HCl (pH 8.0), 10% glycerol, and 10 mM MgCl₂ for 4 h. The enzyme was either assayed immediately for topoisomerase enzyme activity or mixed with an equal volume of 2XSDS loading buffer for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis of lysine acetylation was carried out using mouse monoclonal anti-acetyl lysine antibody (Cell Signaling Technology (Danvers, MA, USA)).