Mouse monoclonal anti ubiquitin antibody p4d1

Five hundred milligrams of 7-day-old wild-type or transgenic seedlings were lysed using a TissueLyser II (Qiagen) and homogenized in 1 ml lysis buffer A [50 mM Tris–HCl pH 7.5, 100 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5% Triton X-100, protease inhibitor mixture (Roche)]. Cell lysate was mildly agitated for 15 min on ice and centrifuged for 15 min at 13 000 g. For lines carrying green fluorescent protein (GFP)-tagged proteins, supernatant was incubated with GFP–Trap magnetic agarose (MA) beads (ChromoTek) for 2 h at 4 °C. Beads were concentrated using a magnetic separation rack. Samples were washed four times in buffer B [50 mM Tris–HCl pH 7.5, 100 mM NaCl, 0.1 mM PMSF, 0.2% Triton X-100, protease inhibitor mixture (Roche)]. Bound proteins were eluted from beads by heating the samples in 30 µl 2× Laemmli buffer for 5 min. Samples were separated by SDS-PAGE and analysed by immunoblotting according to standard protocols. Primary antibodies included mouse monoclonal anti-GFP antibody 3E6 (Invitrogen/Thermo Fisher Scientific), mouse monoclonal anti-ubiquitin antibody P4D1 (Santa Cruz Biotechnology), and polyclonal anti-CHC antibody AS10 690-ALP (Agrisera). Secondary antibodies were obtained from Pierce/Thermo Fisher Scientific: goat anti-rabbit IgG antibody (1858415) and goat anti-mouse IgG antibody (1858413).