Pierce anti ha agarose

C. difficile 630 ΔzmpI harboring pJKP095 was grown to an A_{600 nm} of 1.2 in 1 liter of BHIS liquid medium in the presence of the inducer ATc (100 ng/ml). Cells were harvested by centrifugation at 5000 × g for 10 min at 4 °C and resuspended in phosphate/sucrose buffer to an A_{600 nm} of 40. Purified catalytic domain of endolysin CD27L was added to the cell suspension at 30 μg/ml, and the sample was incubated at 37 °C for 1 h. Protoplasts were pelleted by centrifugation at 6000 × g for 20 min at 4 °C, and the supernatant containing the digested cell wall was collected. Cell wall extracts were concentrated using centrifugal filters (Amicon, Millipore) and resuspended twice in 15 ml of TBS buffer (50 mm Tris-Cl, pH 7.6, 150 mm NaCl). Cell wall extract was then concentrated to a final volume of 400 μl, and 80 μl of Pierce anti-HA-agarose (Thermo Fisher) slurry was added. After an overnight incubation at 4 °C with gentle shaking, resin was pelleted by centrifugation at 12,000 × g for 10 s, washed three times with TBS supplemented with 0.05% Tween 20, and then twice more with TBS. Cell wall-anchored CD2831_R-HA was eluted by addition of 50 mm NaOH to the resin. The eluate was collected by centrifugation at 12,000 × g for 10 s and was neutralized with 1 m Tris-HCl, pH 2.0.

To prepare cell lysate for either IP or Co-IP, cell nuclei were isolated and lysed in IP buffer (25 mM Tris pH 8.0, 0.15 M NaCl, 2.5% glycerol, 0.05% NP-40, 0.05 mM EDTA) with freshly added 1 mM PMSF, 1x protease inhibitor cocktail, 1 mM MgCl₂ and Benzonase (1:500, Novagen). M2 Sepharose beads (Sigma-Aldrich), GFP-Trap® beads (ChromoTek), and Pierce® Anti-HA Agarose (Thermo Fisher Scientific) were used for IP of proteins with FLAG-Tag, GFP-Tag, or HA-Tag, respectively. For endogenous IP, one milligram of nuclear extract was incubated overnight with magnetic beads (Invitrogen Dynabeads) conjugated with respective antibodies. Bound proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) and immunoblot. Primary antibodies for IP assays were listed in Supplementary Table 6.

The surface plasmon resonance experiment was performed on a BIAcore biosensor system (BIAcore T100). In brief, the carboxymethylated surface of the series S sensor chip CM5 (GE Healthcare Bio-Sciences, Sweden) was firstly activated with a mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydro- chloride (EDC) (BIAcore AB). Subsequently, purified hA3G-HA was injected into flow cell 2 (FC2) for immobilization on the sensor surface. To analyze the binding of NS3-His with hA3G-HA, 160 μL of NS3-His [0.3, 0.6, 1.2 or 3 μM in HBS-EP+ buffer (GE Healthcare Bio-Sciences, Sweden)] were injected, followed by flowing the buffer over the chip and a regeneration step with Glycine-HCl (pH 1.5). Differences in resonance spectra (FC2–FC1) were recorded. Data were evaluated using the software Biacore T100 Evaluation (Biacore AB). The HCV NS3-His and hA3G-HA were purified with HisTrap HP (GE Healthcare Bio-Sciences AB, Sweden) and Pierce Anti-HA Agarose (Thermo Scientific, USA) respectively, and desalinated with HisTrap HP Desalting (GE Healthcare Bio-Sciences AB, Sweden) and Zeba Spin Desalting Columns (Thermo scientific, USA) respectively, according to the manufacturer’s operating manuals. The concentration of purified proteins were quantified with Pierce BCA Protein Assay Kit (Thermo Scientific,USA).

Cell lysates harvested using M-PER containing protease inhibitor were centrifuged to collect the supernatant. Pierce™ Anti-HA Agarose (ThermoFisher, Rockford, IL, USA) was added to the supernatant and the mixture was rotated overnight at 4 °C. Following incubation, wash buffer I (50 mM Tris (pH 7.5), 150 mM NaCl, and 0.1% Triton X-100) was used to wash the immunoprecipitates three times. The samples were subsequently subjected to SDS-PAGE and Western blotting analysis.

The immunoprecipitation experiments were performed as described by Du et al. [20 (link)]. In brief, cells were co-transfected with LXRα-HA and SHP-GFP plasmids for 48 h. Then, cells were washed and collected. The supernatant was treated overnight at 4 °C with IgG-beads (Sigma, Missouri, USA), GFP antibody (CST, Danvers, MA, USA) and Pierce^®ANTI-HA agarose (Thermo, Waltham, MA, USA). Then, the fusion protein was eluted for immunoblotting after washing.