Rabbit polyclonal anti gfp antibody

HEK293T cells were transfected with plasmids encoding GFP alone or GFP-tagged WT SrfH (GFP::SrfH) and its truncated forms (amino acids 1 to 140 and 137 to 322) using X-fect transfection reagent (Clontech, Mountain View, CA). For pulldown assays, these transiently transfected HEK293T cells were lysed using 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, and protease inhibitor. The postnuclear supernatant was generated by spinning the lysates at 13,000 rpm at 4°C. Meanwhile, anti-rabbit Dynabeads (Life Technologies) were conditioned by washing three times with PBS and incubation with rabbit polyclonal anti-GFP antibody (Santa Cruz Biotechnologies, Dallas, TX). The antibody was cross-linked to the beads, and the beads were incubated with the transfected HEK293T cell lysates overnight at 4°C. The next day, the beads were washed three times with lysis buffer and resuspended in sample buffer for SDS-PAGE analysis followed by Western blotting using either mouse monoclonal anti-GFP or anti-ERK2 antibodies (Cell Signaling Technologies, Danvers, MA).

Mouse monoclonal anti-nucleolin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, C23 (MS-3), sc-8031), mouse monoclonal anti-β-actin antibody (Sigma, AC-15), mouse monoclonal anti-Flag antibody (ANTI-FLAG M2 Monoclonal Antibody, Sigma, F1804), mouse monoclonal anti-HP1γ antibody (Sigma, MAB3450), mouse monoclonal anti-RNA polymerase I antibody (Santa Cruz Biotechnology, anti-RPA194 antibody (C-1), sc-48385), rabbit polyclonal anti-GFP antibody (Santa Cruz, sc-8334), goat anti-rabbit IgG-HRP (Santa Cruz, sc-2054), goat anti-mouse IgG-HRP (Santa Cruz #sc-2005), Alexa 568-conjugated goat anti-mouse IgG (H+L) (Thermo Fisher Scientific, Waltham, MA, USA, A11004), Alexa 488-conjugated goat anti-mouse IgG (H+L) (Thermo Fisher, A11029), and Alexa 568-conjugated goat anti-rabbit IgG (H+L) (Thermo Fisher, A11036), mouse monoclonal anti-dimethylated histone H3 lys36 antibody (Active Motif, California, USA, MABI0332), and rabbit polyclonal anti-histone H3 antibody (Abcam, Cambridge, UK, ab1791), were purchased. The anti-KDM2A antibody was described previously [19 (link)]. The anti-Fbxl11 (KDM2A) antibody (Abcam, ab99242) was purchased and used to detect the C-terminal half of KDM2A.