Co-immunoprecipitation (Co-IP)
Co-IP was performed as previously described with some changes (Fiil, Qiu, Petersen, Petersen, & Mundy, 2008). 35S:COL3-HA and 35S:COL13-GFP constructs were transformed into EHA105 Agrobacterium cells and then used to generate 35S:COL3-HA and 35S:COL3-HA::COL13-GFP transgenic plants. Proteins were extracted from 18-d-old seedlings. Anti-GFP used in this assay was bought from Abcam (ab290), Anti-HA used in this assay was bought from Sigma (H6908).
A 0.5 g sample of Arabidopsis seedlings were ground in liquid nitrogen, 1 ml (2 volumes) of lysis buffer was added (50 mM Tris pH 8.0, 150 mM NaCl, and 1 mM EDTA, containing 0.01 volume of 1× Protease Inhibitor Cocktail [Sigma]) or protease inhibitors (1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin). It was spun at 16,000 g for 20 min at 4 °C, and the supernatant was transferred to a new microcentrifuge tube. Protein concentration was determined using the Bradford reagent (Bio-Rad) and bovine serum albumin was used as a standard. For immunoprecipitation (IP) reactions, 1 mg of total protein was incubated with 5 μl of polyantiserum (or pre-immune serum) in a total volume of 1 ml of lysis buffer and incubated for 1 h to overnight at 4 °C, and the sample was mixed by inversion. During incubation, a 40 μl wash per reaction of Protein-A-Agarose beads with 1 ml of cold lysis buffer or phosphate-buffered saline (PBS) was used by gentle vortexing and spinning in a microcentrifuge at 12,000 gfor 30 s. The supernatant was carefully removed by aspiration. This process was repeated twice. A 50 μl sample of fresh lysis buffer or PBS was added. The beads were ready to be used. To precipitate the immune complexes, 50 μl of Protein-A-Sepharose (Amersham Pharmacia Biotech) slurry was added and incubated for 2 h to overnight at 4 °C, mixing the sample head-over-tail. The beads were washed in the microcentrifuge tube by resuspension in 700 μl of lysis buffer. It was then centrifuged at 12,000 g for 15–30 s at 4 °C. The effluent was discarded. Steps 7 and 8 were repeated three more times. Next, 40 μl of 2× SDS sample buffer was added to the beads. The beads were gently mixed (no vortexing) to avoid spreading the beads on the column walls. The sample was heated at 95 °C for 5 min to ensure that the Protein-A-Sepharose complex was within the heating well. The sample was then centrifuged at 12,000 g for 30 s. A sample of 10 μl of the eluted immunoprecipitate was loaded on an SDS-PAGE gel (Bio-Rad). For standard western blotting analysis, 50 μg of total protein was loaded. Proteins were electroblotted onto PVDF membranes (Amersham), blocked for at least 1 h at room temperature in PBS-Tween containing 5% (wt/vol) non-fat dried milk. Primary antibodies were added to PBS-Tween containing 5% (wt/vol) non-fat dried milk and incubated for 1 h. Blots were developed using an ECL Kit from Amersham Pharmacia Biotech.