Protein a magnetic beads

Co-immunoprecipitation (Co-IP) assays were performed as described previously (Chen et al., 2010 (link)). Approximately 1–2 mg total proteins extracted from leaves of transgenic Arabidopsis were incubated with an anti- GFP antibody or anti-AtIPK2β antibody overnight at 4 °C, and then Protein A magnetic beads (NEB) were added and incubated for another 2 hours. Subsequently, the beads were washed according to the immunoprecipitation protocol (NEB) and the eluted precipitates were analyzed by western blot using anti-MYC, anti-GFP, or anti-AtIPK2β antibodies.

Cells were lysed in EBC buffer (50 × 10⁻³m Tris pH 7.5, 120 × 10⁻³m NaCl, 0.5% NP‐40) or Triton X‐100 buffer (50 × 10⁻³m Tris pH 7.5, 150 × 10⁻³m NaCl, 1% Triton X‐100) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail sets I and II, Calbiochem). The protein concentrations of whole cell lysates were measured by NanoDrop OneC using the Bio‐Rad protein assay reagent as described previously.^[55] Equal amounts of whole cell lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotted with indicated antibodies. For immunoprecipitation analysis, unless specified, 1000 µg lysates were incubated with the indicated antibody (1–2 µg) for 3–4 h at 4 °C followed by 1 h incubation with 10 µL Protein A magnetic beads (New England Biolabs). Or, 1000 µg lysates containing tagged molecules were incubated with agarose‐bead‐coupled antibodies for the specific tag for 3–4 h at 4 °C. For endogenous IPs, incubation of cell lysates with antibodies was extended to overnight. The recovered immunocomplexes were washed 5 times with NETN buffer (20 × 10⁻³m Tris, pH 8.0, 100 × 10⁻³m NaCl, 1 × 10⁻³m ethylenediaminetetraacetic acid (EDTA), and 0.5% NP‐40) before being resolved by SDS‐PAGE and immunoblotted with indicated antibodies.