Iblot protocol

Lysates and extracts were prepared as previously described^{48 (link)}. Immunoprecipitation of Spo11-oligo complexes was performed using 5 μg of mouse monoclonal anti-flag M2 antibody (Sigma). Precipitated Spo11-oligo complexes were end-labeled in NEBuffer 4 (New England Biolabs) containing 3–10 μCi of [α-³²P]dCTP and terminal deoxynucleotidyl transferase (TdT)^{48 (link)}. Twenty-five μl of reaction mixture was added to the beads, mixed, and incubated at 37°C for 1–2 hr. Spo11-oligo complexes were eluted by adding 25 μl of NUPAGE® loading buffer (diluted to 2× and supplemented with 83.3 mM dithiothreitol) (Invitrogen) and boiling for 5 min. End-labeled Spo11-oligo complexes were separated on a Novex® 4–12% gradient denaturing polyacrylamide gel (Invitrogen) then transferred onto PVDF membrane using the iBlot protocol (Invitrogen) and visualized by phosphorimager. Blots were probed with mouse monoclonal anti-flag M2 conjugated to horseradish peroxidase (Sigma). Chemiluminescent detection was performed according to the manufacturer's instructions (ECL+ or ECL Prime, Amersham). Protein quantity was estimated by separating 1 μl of extract on a Novex® 4–12% gradient denaturing polyacrylamide gel and staining with Coomassie Brilliant Blue.