Phosphorylated Antibody Detection Protocol

The pAb506P rabbit IgG was generated to a 21-amino acid phosphopeptide (TVGCCS*LRVEHINLHPELKKC, in which phosphoserine 506 is indicated by *), as previously described [1 (link)]. Goat anti-topo I IgG, which recognizes the topo I C-terminus, and mouse monoclonal anti-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-tubulin was purchased from Novus Biologicals (Littleton, CO). The secondary antibodies were goat anti-rabbit-horseradish peroxidase (HRP), goat anti-mouse-HRP, and donkey anti-goat-HRP (Santa Cruz Biotechnology). For post-immunoprecipitation Westerns, the primary antibody was detected using Pierce Clean-Blot IP Detection Reagent (Thermo Scientific, Waltham, MA), which recognizes only whole, intact IgG and not the dissociated light or heavy chain subunits.