Gfp trap m magnetic beads

Protein fusion constructs were transiently overexpressed in N. benthamiana using Agrobacterium-mediated expression. Leaf disks were collected 48 h after infiltration. Samples were ground in liquid nitrogen, and tissue was resuspended in 500 µL GTEN extraction buffer (as above), gently vortexed, placed on ice for 10 min, and centrifuged at 13,000 × g for 10 min at 4 °C. For input samples, 40 µL of sample was removed, mixed with 40 µL 2× SDS-PAGE sample buffer, boiled at 95 °C for 10 min, and stored at −20 °C for western blot analysis. The remaining sample extract was incubated with 20 µL GFP-Trap-M magnetic beads (Chromotek; beads were prewashed three times with 500 µL ice cold wash buffer [GTEN with 1 mM PMSF]) on a rotary mixer for 1 h at 4 °C. Beads were magnetically separated from the sample supernatant and washed three times with 500 µL ice cold wash buffer; then, they were resuspended in 50 µL 2× SDS-PAGE sample buffer and boiled at 95 °C for 10 min. The resulting samples were separated by SDS-PAGE and analyzed by immunoblotting as above.

Cells were lysed in 300 µl lysis buffer (10 mM Tris HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 10 mM Na₂MoO₄, 0.5% NP-40) and DNA was sheared by sonication for 3 s. The lysate was cleared by centrifugation at 12,000 g for 15 min and protein amount was normalized to 1.25 µg/µl using dilution buffer (10 mM Tris HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 10 mM Na₂MO₄). The lysate was incubated with 20 μl of GFP-Trap-M magnetic beads (ChromoTek, Germany) for 90 min at 4°C. Beads were washed thrice with dilution buffer and twice with MS wash buffer (20 mM Tris HCl pH 7.5, 150 mM NaCl) and then snap-frozen in liquid nitrogen and stored at −80°C until further processing. Beads were re-suspended in 50 μl 8 M urea, 50 mM Tris HCl pH 8.5, reduced with 10 mM DTT for 30 min and alkylated with 40 mM chloroacetamide for 20 min at 22°C. Urea was diluted to a final concentration of 2 M with 25 mM Tris HCl pH 8.5, 10% acetonitrile and proteins were digested with trypsin/lysC mix (mass spec grade, Promega, Madison, WI) overnight at 24°C. Acidified peptides (0.1% trifluoroacetic acid final concentration) were desalted and fractionated on combined C18/strong cation exchange StageTips. Peptides were dried and resolved in 1% acetonitrile, 0.1% formic acid.