GST-fusion proteins were used in pull-down assays with a pool of Lambda DNA restriction fragments. The assay was performed as described previously (11,12). Restriction fragments were filled in with [α-32P]dATP. Labeled DNA (0.8 μg) was incubated with 50 ng of GST-fusion protein bound to glutathione–agarose beads for 1 h at 4°C in Lambda DNA-binding buffer [20 mM HEPES (pH 7.6), 1 mM EDTA (pH 8), 10 mM (NH4)2SO4, 0.2% Tween-20, 1 mM DTT, 25 μg/ml BSA and 25 μg/ml poly(dI–dC)] plus varying amounts of KCl, as indicated in the text. The beads were washed three times with Lambda DNA-binding buffer minus DTT, BSA and poly(dI–dC). Bound DNA was eluted by boiling in Formamide loading buffer (90% formamide, 1× TBE, 0.04% bromophenol blue and 0.04% xylene cyanol), separated on a 6% sequencing gel and visualized by autoradiography.
