Gfp trap beads

Large-scale immunoprecipitation assays for LC-MS/MS analysis were performed as previously described with several modifications [67 (link)]. Two grams of N. benthamiana leaf tissues were collected at 2 days after infiltration with A. tumefaciens and frozen in liquid nitrogen. The protein extracts were homogenized in protein extraction buffer (100 mM Tris-HCl pH8, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 10 mM DTT, 2 mM PMSF, 10 mM NaF, 10 mM Na₂MoO₄, 2 mM NaVO₃, 1%(v/v) NP-40, 1%(v/v) plant protease inhibitor cocktail (Sigma). The protein extracts were cleaned by 2 rounds of 10 min x 15, 000 g centrifugation to remove the tissue debris. 30 μL GFP-trap beads (ChromoTek, Germany) or ANTI-FLAG M2 Affinity Agarose Gel (Sigma) was added to the clean protein extract and incubated for one hour at 4°C with an end-to-end slow but constant rotation. GFP-trap beads or ANTI-FLAG beads were washed four times with 1 mL cold wash buffer (100 mM Tris-HCl pH 8, 150 mM NaCl, 10% glycerol, 2 mM DTT, 10 mM NaF, 10 mM Na₂MoO₄, 2 mM NaVO₃, 1%(v/v) plant protease inhibitor cocktail (Sigma), 0.5%(v/v) NP-40 for GFP-trap beads, no NP-40 for anti-FLAG M2 beads) and the washed beads were subjected to Mass Spectrometric analysis as previously described to identify interacting proteins [66 (link)] and phosphorylated peptides [68 (link)].

Endogenous immunoprecipitation was performed using DSP (Lomant’s Reagent) (Thermo Scientific) on intact cells at the indicated concentrations for 30 min on ice. Tagged proteins were immunoprecipitated with either GFP-Trap beads or anti-FLAG M2 affinity gel (Sigma). See Supplemental Experimental Procedures for more information.

N. benthamiana leaves (four-weeks-old) were co-infiltrated by agro-infiltration. After 48 hours, the leaf tissue was collected, quick-frozen with liquid nitrogen and ground into powder. Total proteins were extracted using cold IP buffer (40 mM Tris-HCl (pH = 7.5), 100 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 1% glycerol with 1 mM DTT, 1 mM PMSF) for 20 min on ice and centrifuged twice at highest speed for 10 min at 4°C. Then, 60μl was used as input control for western blot and the remaining supernatant was transferred to a new microcentrifuge tube, where it was incubated with 5μl FLAG-trap beads or GFP-trap beads (Sigma-Aldrich, USA) at 4°C for approximately 2 h. The supernatant was then removed and the beads were washed at least three times in ice-cold IP buffer to remove the non-specifically adsorbed proteins. Subsequently, all protein samples were boiled for 10 minutes, briefly centrifuged and then separated on a 12% SDS-PAGE gel followed by western blotting analysis.