Mouse anti strep tag 2 monoclonal 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.

Cells grown in LB to the mid-exponential phase before and after induction of IPTG at proper concentrations for 10 h were collected and lysed by sonication with a Xinzhi Sonifier (JY92-IIDN, NingBo, China) at maximum output at 0°C until no whole bacterial cells were visible, and the protein extracts were collected after centrifugation at 3,000 g for 10 min. The extracts were mixed with 4× SDS-loading buffer, denatured for 10 min at 100°C, and resolved by 12% SDS-PAGE gel. The resolved proteins were directly stained either with Coomassie Brilliant Blue R-250 or with 3,3′,5,5′-tetramethylbenzidine for heme-staining analysis (65 (link)) or subject to Western blotting. Protein transfer onto the PVDF membrane (GE-Healthcare, Shanghai, China) was carried out for 1 h at 60 V in a Criterion blotter (Bio-Rad, Shanghai, China) with Tris-Glycine transfer buffer. To detect proteins with Strep-tag II, the blotting membrane was probed with the primary antibody mouse anti-Strep-tag II monoclonal antibody (1:5,000) (Abbkine, Shanghai, China) and then the second antibody goat anti-mouse IgG-HRP (1:100,000) (Beyotime, Beijing, China). To detect biotinylated proteins, HPR-streptavidin was used as the probe. The blots were finally developed by chemiluminescence detection with SuperSignal West Dura Extended Duration Substrate kit (Invitrogen, Shanghai, China) and visualized with the CLiNX system.