Anti flag agarose a2220

PBS-washed S2 cells or dechorionated embryos were homogenized in ice-cold lysis buffer (HA immunoprecipitations: 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.75% NP-40, 0.1 mM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.7 µg/ml pepstatin; FLAG immunoprecipitations: 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, pH 8, 5% glycerol, 1% Triton X-100, 40 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.7 µg/ml pepstatin). Cellular debris was then removed by centrifugation (17,000 g for 10 min at 4°C), and embryo or S2 cell lysates containing 500 µg to 1.5 mg of total proteins were precleared for 90 min under agitation with 15 µl of a suspension of protein G Sepharose beads (50% in lysis buffer; GE Healthcare). Supernatants were then incubated with 0.5 µg of the anti-HA antibody (clone 16B12; BioLegend) or with 20 μl of anti-FLAG-agarose (A2220; Sigma-Aldrich) for 1.5 h at 4°C under agitation. HA immunocomplexes were further incubated (1 h at 4°C) with 15 µl protein G Sepharose beads. Beads were harvested by centrifugation and washed five times with lysis buffer. Proteins were eluted with Laemmli buffer.

Rabbit polyclonal antibodies were raised against purified, recombinant Ell1, Eaf1 and Ebp1 (Bio-Synthesis, Inc.) and purified using Melon™ Gel (ThermoFisher Scientific) according to the manufacturer's instructions. Anti-Pol II (4H8) was obtained from Abcam (ab5408). Mouse monoclonal anti-Myc (M4439) and anti-HA (H9658), and rabbit anti-FLAG (F7425) antibodies, as well as anti-FLAG agarose (A2220) and anti-Myc agarose (E6654) beads, were from SIGMA. Alexa Fluor 680-goat anti-mouse and Rabbit IgG DyLight™ 800 antibodies were from Invitrogen (A21058) and Rockland Antibodies (611-745-127), respectively.