Protein pull-down assay

Binding reactions (20 μl) were carried out in 25 mM Tris-HCl pH 7.5, 10% glycerol, 100 mM NaCl, 2 mM DTT, 5 mM EDTA, 200 μg/ml BSA, 0.1% Triton X-100 with 25 nM immobilized DNA and the indicated concentrations of MutLγ. Complexes were assembled for 1 hour at 4°C on a rotating wheel. The beads were washed three times with 200 μl binding buffer without BSA, resuspended in 1× Laemmli sample buffer, boiled for 5 minutes and loaded on 4–12% Bis-Tris NuPAGE gels in MOPS running buffer. Proteins were detected by silver staining or anti-Flag western blotting.