Mouse anti ha igg

Cell lysate collected from C6/36 cells transfected with the plasmid pAC5.1-HA-C189 was centrifuged using a Microfuge 22R microcentrifuge (Beckman Coulter), and PNS was harvested for further magnetic immunoisolation by using a Dynabeads Protein G Immunoprecipitation Kit (Invitrogen). To prepare anti-HA Dynabeads, we added 50 μl of them coated with protein G into the 8-tube Magnetic Separation Rack to remove the supernatant. Then, 100 μl antibody binding and washing buffer containing 10 μg mouse anti-HA IgG (Sigma) was added into the tube and gently shaken for 1 h. After another wash with antibody binding and washing buffer and centrifugation to remove the supernatant, prepared anti-HA Dynabeads were collected and subsequently mixed with PNS. After shaking at RT for 1 h, the mixture was transferred to an 8-tube Magnetic Separation Rack and washed three times with 200 μl of washing buffer, followed by adding100 μl of fresh washing buffer to resuspend the mixture and then moved into a new collection tube. For protein analysis, a mixture of 30 μl 1x Laemmli sample buffer was heated in a water bath at 95°C for 5 min. Protein was then collected from the supernatant from the 8-tube Magnetic Separation Rack and subjected to Western blot analysis. For RNA detection, samples were RNA-extracted via a GENEzol TriRNA Pure Kit (Geneaid Biotech).

The cell lysates were obtained as previously described (Irazoki et al., 2016 (link)). Cultures of S. enterica ΔrecA ΔcheW harboring the plasmids encoding the corresponding tagged proteins were used, and the gene overexpression was induced by the addition of 1 mM IPTG. As a control, cell lysates of S. enterica ΔrecA ΔcheW containing the pUA1108 overexpression vector (Mayola et al., 2014 (link)) were processed according to the same procedure.
The immunoprecipitation assays were performed using Pure Proteome Protein A magnetic beads (Millipore) coated with either mouse anti-FLAG IgG (Sigma-Aldrich) or mouse anti-HA IgG (Sigma-Aldrich) monoclonal primary antibodies, following the manufacturer’s instructions. Cell lysates were mixed at a molecular ratio of 1:1 and incubated at 30°C for 1 h without shaking to allow protein–protein interaction.
As a final step, the samples were separated by SDS-PAGE on a 15% polyacrylamide gel and analyzed by Western blotting using a horseradish-peroxidase (HRP)-coupled anti-mouse antibody (Acris). The membranes were developed using a HRP chemoluminiscent substrate (Luminata ForteTM Western HRP substrate, Millipore) following the manufacturer’s instructions. The membranes were imaged using a ChemiDocTM XRS+ system (Bio-Rad).