Ab94010

EMSAs were performed as previously described^{35 (link)}. Flag-tagged proteins were purified from RNaseIII^−/− cells at 24 hpt in lysis buffer (15 mM Tris/HCl pH 7.5, 150 mM NaCl, 15 mM MgCl₂, and 1% (v/v) TritonX-100) supplemented with 1x protease inhibitor cocktail (Roche®). Cell lysates were incubated with anti-Flag affinity resin (Sigma A2220) at 4 °C overnight, followed by four washes at 5 min each with lysis buffer. Bound protein was eluted with 300 μg/ml Flag-peptide (Sigma F3290) in 20 mM Tris/HCl pH 8.0, 100 mM KCl, and 0.2 mM EDTA. Alternatively, a fragment of recombinant human Drosha protein was purchased from Abcam (ab94010) and 0.25–1 μg of this purified protein was used for EMSAs. Radioactively labeled RNA was in vitro transcribed from a PCR-amplified template using the MAXIscript kit (Ambion®). For complex formation of protein and RNA, protein eluate or recombinant protein was incubated with radio-labeled RNA (~100,000 cpm per reaction) for 30 min at room temperature in 20 mM Tris/HCl pH 8.0, 100 mM KCl, and 0.2 mM EDTA, supplemented with 0.1 μg/μl BSA and 1 mM DTT. Reactions were run on a 6% polyacrylamide gel.