![]() Radiolabeled RNAs were annealed by heating to 95 ☌, snap cooling to 4 ☌, addition of binding buffer, followed by equilibration to rt over at least 20 min. Protein-RNA binding was carried out with indicated amounts of protein and 5′ 32P labeled RNA in binding buffer (50 mM Tris-HCl, 100 mM KCl, 5 mM MgCl 2, 10 mM β-ME, 0.1% IGEPAL CA-630), with yeast tRNA (Roche) at a concentration of 1.25 μg/μL. Recombinant LSD1 (ab80379) was purchased from AbCam. Protein purity was assessed by SDS-PAGE and Western blotting ( SI Figure 1 antibodies listed in Table S3). Combined Histrap fractions were then incubated with prewashed M2 beads, and further purified following the procedure for FLAG purification. For dual tagged complexes (FLAG-EZH2–6×HIS-EED and PRC2 3m), purification initially followed the procedure for 6×His tag purification. Combined peak fractions were further purified by gel filtration followed by spin concentration. Cleared extracts were loaded onto a Histrap FF column (GE Healthcare) and eluted with a 50–500 mM imidazole gradient. For 6×His-EED, pellets were resuspended in Histrap buffer (as for BC300, but including 50 mM imidazole), lysed by sonication, and extracts cleared by centrifugation. Where necessary, proteins were further purified by gel filtration using a HiLoad 16/600 Superdex 200 column (GE healthcare), followed by concentration using Amicon ultra 10 kDa MWCO spin concentrators (Millipore). Beads were eluted with 0.2 mg/mL 3xFLAG peptide (Sigma), and desalted using Zeba spin desalting columns (Pierce). Beads were then pelleted, briefly washed 3× with BC2000 (as above, but 2 M KCl), and once with BC300. Cleared extracts were incubated with prewashed M2 beads (Sigma) for 4 h at 4 ☌. For FLAG tagged proteins (FLAG-EZH2 and FLAG-EZH2-SUZ12), pellets were resuspended in BC300 buffer (20 mM HEPES, 300 mM KCl, 0.2 mM EDTA, 10% glycerol, 1 mM DTT, 0.2 mM PMSF, and complete protease inhibitors (Roche)), lysed using sonication, and extracts cleared by centrifugation. High Five cells were harvested 48 h after infection and purified according to tag. For dimers and trimers, the complexes were expressed by coinfection and purified together as a single unit, rather than mixing prepurified monomers. Proteins were expressed in High Five insect cells using baculoviral expression vectors. Transfer plasmid pFastBac-FLAG-EZH2 was a generous gift from Prof. Kristian Helin, University of Copenhagen. ![]() Baculovirus for 6xHIS-EED and SUZ12 were a generous gift from Prof. ![]()
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