Dynabeads protein a magnetic beads

Immunoprecipitations were performed using Dynabeads protein A magnetic beads coupled to polyclonal GFP antibodies (ab290, Abcam) using the crosslinker, Bis(sulfosuccinimidyl)suberate (BS3), according to manufactures instructions (Thermo Scientific). For MS-analysis: Coupled beads were incubated with XRCC1-EYFP overexpressed HeLa S3 cell lysates of untreated cells or cells treated with H₂O₂ under gentle rotation at 4 °C overnight followed by washing 3 times with 10 mM Tris-HCl pH 7.5, 50 mM KCl before elution. Co-immunoprecipitation analysis: Beads were incubated with lysates from HeLa S3 cells transfected with YFP-MD wild type and mutant plasmid construct. The cells were pretreated for 1 hour at 37 °C with 0.25% DMSO (mock) or 10 µM PARP inhibitor (PJ34). Whole cell extracts were prepared as described above, with exception of PJ34, that were added to buffer I to a concentration of 10 µM prior to lysis. Beads were washed three times in 10 mM Tris-HCl pH 7.5 containing either 100 mM or 1 M NaCl. Elution: Immunoprecipitated proteins were eluted in LDS loading buffer (Invitrogen) containing 100 mM DTT by heating the beads for 10 minutes at 70 °C and separated on a NuPAGE 4–12% Bis-Tris protein gel (Invitrogen) using MOPS SDS running buffer (Invitrogen). Proteins were blotted to polyvinylidene fluoride (PVDF) membranes (Immobilon, Millipore).

Endogenous XRCC1 from HeLa cells was immunoprecipitated over night at 4 °C using Dynabeads protein A magnetic beads coupled to monoclonal XRCC1 antibodies (ab1838, Abcam). The beads were washed three times with a 10 mM Tris-HCl pH 8 and 50 mM KCl buffer, and divided in two equal parts prior to resuspension in phosphatase buffer (10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl₂, 1 mM DTT, and 0.1 mM ZnCl₂). Excess of calf intestinal phosphatase (CIP, Biolabs) was added to one of the tubes following incubation for 1 hour at 37 °C. Proteins were eluted from beads by overnight incubation in Destreak solution containing 1% IPG buffer pH 4–7 (GE Healthcare). Eluates were collected and submitted to 2D-PAGE as described above.