Fig. 2:

Analysis of different steps during chromatin immunoprecipitation using ERα antibody of uterine extracts. Uterine tissues were collected following injection of oil (as vehicle) or E2 (100 ng/mouse) for 24 h in ovariectomized mice. Uterine tissues extracts without cross-linking (A), following the cross-linking using 1% formaldehyde (B), following the cross-linking and reversal by heat treatment (C) or following the cross-linking and fragmentation of the chromatin by sonication (D) were analyzed by Western blotting (WB) using ERα antibody. Arrows indicates the position of un-complexed ERα band, while a vertical line in (B) represents macromolecular association of ERα. In panel C, the asterisk (*) denotes a band exhibiting reversal of ERα association. Results show that ERα-associated high molecular complex with retarded migration in SDS-PAGE gel can be reversed by prolonged heat treatment (B vs. C). Additionally, the application of sonication following fixation does not cause any detrimental effect to ERα protein for its normal detection after WB (D) and IP/WB (F), but eliminates higher molecular retarded bands as seen in (B). Furthermore, following the sonication step, DNA fragments were recovered by phenol:chloroform extraction and alcohol precipitation, and then analyzed by 2% agarose gel (E). M = 1 kb marker.