Anti flag agarose affinity resin

Native complexes were purified essentially as previously described (35 (link)). Briefly, nuclear extracts were prepared from 2.5.10⁹ cells and precleared with CL6B sepharose beads. FLAG immunoprecipitations with anti-FLAG agarose affinity resin (Sigma; clone M2) were performed followed by two elutions with 3xFLAG peptide in elution buffer (20 mM Hepes–KOH [pH 7.9], 10% glycerol, 150 mM KCl, 0.1% Tween-20, 1 mM DTT, 1 mM PMSF, 2 mg/ml leupeptin, 5 mg/ml, 2 mg/ml pepstatin, 10 mM sodium butyrate, and 10 mM β-glycerophosphate) with 200 μg/ml 3xFLAG peptide (Sigma). Then, STREP immunoprecipitations with Strep-tactin sepharose beads (Cedarlane) were performed followed by two elutions with elution buffer supplemented with 4 mM biotin. Typically, 20 μl of the first Strep elution was loaded on SDS-Nu-PAGE 4 to 12% Bis–Tris gels (Invitrogen) and analyzed via silver staining. Fractions were then analyzed by mass spectrometry (MS) and Western blotting. For purification after inducing DNA damage, cells were treated with 50 ng/ml of neocarzinostatin for 3 h before being collected to prepare the nuclear extract.