Structure of the chromatin remodelling enzyme Chd1 bound to a ubiquitinylated nucleosome

Ramasubramanian Sundaramoorthy; Amanda L. Hughes; Hassane El-Mkami; David Norman; Tom Owen-Hughes

Proteins were re-suspended in 50mM ammonium bicarbonate pH 8 and treated with 2mM TCEP for 1 hour. Ellman's reagent was used to ascertain the concentration of free sulfhydryls, and xH2b and ubiquitin were combined at equimolar ratios, as defined by the Ellman's assay, and diluted with 50mM ammonium bicarbonate to 200-250uM each protein. The proteins were crosslinked at room temperature with four hourly additions of ¾ molar ratio of 1,3 dichloroacetone (freshly prepared in DMF). An equal volume of denaturing buffer (7M Guanidine HCl, 350mM sodium chloride, 25mM Tris pH7.5) was added to the reactions, which were purified using HisPur cobalt resin, preequilibrated in denaturing buffer. The His-TEV-Ub-xH2B crosslinked product was eluted with 350mM imidazole and dialysed into SAUDE200 buffer (7M Urea, 20mM sodium acetate, 200mM sodium chloride, 1mM EDTA, 5mM 2mercaptoethanol) overnight. The ubiquitinated histone was further purified over a cation exchange column, as before, and fractions were dialysed into water with 2mM 2-mercaptoethanol and lyophilised for storage.