Fig. 2 . Production of polyclonal antibodies against macroH2A. (A) MacroH2A consists of an N-terminal region (red) that is 64% identical to canonical H2As followed by a large C-terminal nonhistone domain (NHD) of unknown function (green). The dark green box labeled 1.2 indicates the position of one of two potential leucine zipper domains generated by alternative splicing. A PacI site was introduced into the macroH2A.2 cDNA at the junction of the histone and nonhistone domains (black triangle), allowing fusion of the nonhistone domain to GST. The GST-NHD fusion contains a thrombin cleavage site (open triangle) at the GST-NHD junction, allowing proteolytic cleavage to yield free NHD protein. (B) Recombinant NHD expression in bacterial cells. Lanes 1 and 2: Coomassie Blue-stained total bacterial protein from uninduced (lane 1) and IPTG-induced cells (lane 2) containing a GST-NHD expression plasmid. Lane 3: GST-NHD fusion protein purified from bacterial cells on a glutathione column eluted with reduced glutathione elution. Lanes 4 and 5: Purified NHD protein was eluted from a glutathione resin charged with GST-NHD by cleaving with thrombin. This allowed free NHD fusion protein to be eluted in two washes (samples of these supernatants are shown). (C) NHD protein from the same sample loaded in lane 4 was used to inoculate a rabbit that produced antibodies that recognize recombinant GST-NHD protein (lane 1) and endogenous macroH2A in total ES cell protein (lane 2) by immunoblotting.