Anti gfp from mouse igg1κ

FRET 2in1 destination vectors containing monomeric enhanced green fluorescent protein (mEGFP) and mCherry (pFRETgc-2in1) were used to transiently express recombinant proteins in N. benthamiana for co-IP analysis (28 (link), 35 (link)). Leaf material (150 to 600 mg) was harvested at 3 d postinfiltration and homogenized after freezing in liquid nitrogen. Lysis buffer (25 mM Tris pH 8.0, 150 mM NaCl, 1% Nonidet P-40, and 0.5% Na-deoxycholate) supplemented with protease inhibitor mixture and 2 mM DTT was added and incubated for 1 h at 4 °C with mild rotation. After centrifugation, the supernatant was mixed with 20 to 25 µL of RFP beads (RFP-trap; Chromotek) and then incubated for 1 h at 4 °C with mild rotation. Beads were collected by centrifugation, transferred onto spin columns, and rinsed twice with lysis buffer, followed by six washes with wash buffer (25 mM Tris pH 8.0 and 150 mM NaCl). (Co-) Immunoprecipitated proteins were eluted with 2× Laemmli buffer (+ 3.5% β-mercaptoethanol) and then heated at 65 °C for 15 min (membrane proteins) or at 95 °C for 5 min (soluble proteins). Proteins were separated by SDS-PAGE and detected by Western blot analysis (anti-RFP from mouse [Chromotek, 1:2,500], anti-GFP from mouse IgG1κ [Roche, 1:1,000], and anti-mouse IgG [Fc-specific] produced in goat [Sigma-Aldrich, 1:10,000]).

Here 3 g of Arabidopsis seedlings were harvested after 10 d under continuous light. Cells were lysed by mortar grinding in liquid nitrogen and thawed in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100; 1.43 mL/g) supplemented with protease inhibitor mixture (cOmplete EDTA-free; Roche). Cell debris was removed by centrifugation and filtration through two layers of Miracloth. Then 2.5 mL of supernatant was mixed with 2 mL of lysis buffer and incubated with anti-GFP beads (25 µL, GFP-trap; Chromotek) for 2 h at 4 °C under mild rotation. Beads were collected by centrifugation, transferred onto spin columns, and washed six times with washing buffer (50 mM Tris pH 7.5, 150 mM NaCl, and 0.5% Triton X-100) supplemented with protease inhibitor mixture. (Co-) Immunoprecipitated proteins were eluted with 2× Laemmli buffer (+ 3% β-mercaptoethanol) at 80 °C for 5 min, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and detected by Western blot analysis (anti-HA peroxidase from rat IgG1 [Roche, 1:1,000], anti-GFP from mouse IgG1κ [Roche, 1:1,000], and anti-mouse IgG [Fc-specific] produced in goat [Sigma-Aldrich, 1:10,000]).