Total bone marrow cells (2 × 106 cells per ml) or purified monocytes (0.5 × 106 cells per ml) were suspended in complete RPMI-1640 (supplemented with 10% (vol/vol) dialysed FBS (Thermo Fisher Scie
After completion of the gradient the refolded nucleosomes were dialysed two times against no-salt buffer (10 mM TRIS pH 7.5, 1 mM EDTA, 1 mM DTT) CD analysis of the H2A/B dimers. CD spectra were recorded using a Chirascan CD-spectrophotometer (Applied Photophysics). H2A/B dimers were ...
Eluates were dialysed with 10 kDa cut-off dialysis tubing against Tris-HCl buffer (50 mM Tris-HCl, pH 7,5, 500 mM NaCl and 1 mM DTT) overnight at 4°C. S2 cells transfected with plasmid encoding either GFP-Su(var)2-10-PA, GFP-tagged truncated Wde including predicted SIM-s 3 to...
the BMPRII ECD5. Briefly, total periplasmic proteins were extracted following the pET System Manual (Novagen) and His-tagged fusion proteins were purified on a 5 ml nickel-nitrilotriacetic acid column (GE Healthcare). Fractions containing the fusion protein were pooled, dialysed into Tris ...
(250 mM KCl, 10 mM Tris pH 7.5, 1 mM EDTA, 1 mM DTT over 36 h) for large-scale reconstitution or against a series of step gradients for micro-scale reconstitution as described12. After completion of the gradient the refolded nucleosomes were dialysed two times against no-...