Monoclonal anti flag antibody clone m2

Rec12-oligonucleotide complexes in ctp1⁺ and ctp1 mutants were assayed as described (5 (link)). Briefly, meiosis was induced and cells were harvested hourly for 6 h. DNA content was analyzed by flow cytometry (unpublished data similar to those in Supplementary Figure S2). The cells were lysed by beating with glass beads in ice-cold 10% trichloroacetic acid. The precipitated proteins were solubilized in sodium dodecyl sulfate (SDS) extraction buffer, diluted in 2x immunoprecipitation (IP) buffer and incubated overnight with monoclonal anti-FLAG antibody (clone M2, Sigma-Aldrich) pre-bound to magnetic protein G-agarose beads (Dynabeads, Invitrogen). After washing of the beads, the bound oligonucleotides were labeled using terminal deoxynucleotidyltransferase (TdT) and [α-³²P] dCTP. The complexes were washed and eluted by boiling. The eluted Rec12-oligos were separated by SDS-PAGE and transferred to Immobilon-P membrane (Millipore). The DNA was detected by exposure to X-ray film, and the Rec12-FLAG protein was detected by immunoblotting using anti-FLAG antibody conjugated to horseradish peroxidase (clone M2, Sigma) and an enhanced chemiluminescence detection kit (Supersignal, WestPico-Pierce).