Comparison of Various Epitope-tagged RRM2B Proteins and Purification of Flag-tagged RRM2B Associated Complexes from 293 T-REx Cells.
(A) Expression of untagged or epitope-tagged RRM2B proteins in 293 T-REx cells was induced with doxycycline (DOX) for 24 hours. Whole cell lysates (WCL) were precipitated with antibodies to Flag or HA beads as well as Strep-Tactin resins. (B) Same cell lysates in (A) were precipitated with RRM2B antibodies or control rabbit IgG. N-: epitope-tagged at the N-terminus; C-: epitope-tagged at the C-terminus (C) Large-scale immunoprecipitation (IP) was performed using whole cell lysates (WCL) from 293 T-REx-Flag-RRM2B cells (F) or control cells expressing untagged RRM2B (U) treated with DOX for 24 hours. A small fraction from each step during purification was saved for analysis. Sup: supernatant following IP; Beads: complexes pulled-down by Flag beads before elution and Elution: urea-eluted complexes. Denatured whole cell lysates and immune complexes electrophoretically separated on gels were transferred to membranes and blotted with antibodies to Flag, HA and RR subunits. (D) A small fraction of protein complexes after urea-elution was separated on a gel by electrophoresis, stained with SYPRO Ruby, visualized using UV-transilluminator and photographed by CCD camera.