Monoclonal anti flag m2 primary antibody

For Western blotting, two colonies were collected per sample, resuspended in buffer (20 mM Tris pH 7.5, 200 mM NaCl, 5 mM EDTA and protease inhibitor [cOmplete protease inhibitor cocktail tablets; Sigma Aldrich]), disrupted on a bead beater (Mini beadbeater, Biospec Products; 4°C, maximum speed for 1 min 15 s), and the total protein concentration of the samples was determined by Bradford assay (Bradford, 1976 (link)) with Protein Assay Dye Reagent Concentrate (Bio-Rad). Samples were normalized by total protein content and run in a 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel for protein separation. Proteins were then transferred to an Immobilon-P transfer membrane (Millipore) using a Trans-Blot Turbo Transfer system (Bio-Rad). After blocking the membrane at room temperature for 2 hr with TBS-T buffer (24.2 g Trizma, 87.6 g NaCl in 1 l H₂O, pH 7.5, 0.1% (v/v) Tween-20) containing 5% (w/v) milk, the membrane was incubated at 4°C o/n with the monoclonal anti-flag M2 primary antibody (Sigma Aldrich) in TBS-T buffer containing 5% (w/v) milk, washed with TBS-T buffer, incubated at RT for 1 hr with α-mouse secondary antibody in TBS-T buffer and again washed with TBS-T buffer. BipA-flag was detected by addition of SuperSignal West Pico Chemiluminiscence substrate (Thermo Scientific) and detection of the signal using a LAS4000 (Fujifilm).

A 50 µg of S. mutans protein lysate was separated via 14% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and transferred to a nitrocellulose blotting membrane with 0.45 μm pore size (transfer buffer: 25 mM Tris, 200 mM glycine, and 20% [vol/vol] methanol) for 75 min at 100 V. The membrane was blocked in PBS containing 3% (wt/vol) skim milk (Sigma) for 30 min at room temperature and subsequently incubated with primary antibodies overnight at 4 °C. Monoclonal anti-FLAG M2 primary antibody (Sigma # F3165) was used at a 1:2000 dilution, while the HA Epitope Tag primary antibody (ThermoFisher # 26183) was used at a 1:4000 dilution in PBS with 3% (wt/vol) skim milk. The membrane was washed with PBS, washed twice with tris-buffered saline containing 0.1% (vol/vol) Tween-20 (TBST), followed by a final wash in PBS. Afterward, the membrane was incubated with anti-IgG secondary antibody (Sigma # 12-349) at a 1:2000 dilution in TBST for 1–2 h at room temperature. Washing steps were repeated as described above. For signal detection, the membrane was incubated with Amersham ECL Prime Western Blotting Reagent (GE Healthcare) and imaged using an ImageQuant LAS 4000 (GE Healthcare). All blots presented as part of the same series were derived from the same experiment and were processed in parallel. Original blots are provided in Supplementary Information.