Figure 2

Purification of 53BP1 from HeLa nuclear extracts. (A) Phosphocellulose chromatography of HeLa cell nuclear extracts. Nuclear extracts were fractionated by p11 phosphocellulose chromatography. Bound proteins were eluted by increasing concentrations of KCl and analyzed for 53BP1 by western blotting. (B) Gel filtration chromatography of 53BP1 and SRC3. The 0.1 M phosphocellulosose fraction was concentrated and purified by gel filtration chromatography. Fractions were collected and assayed by western blotting using α53BP1 and αSRC3 antibodies. (D) Affinity purification of 53BP1. The 0.1 M 53BP1 gel filtration fractions containing SRC3 and 53BP1 were pooled and passed through an α53BP1 affinity column. Bound proteins were eluted with 100 mM glycine (pH 3.0), fractions were collected and aliquots were analyzed using the specific antibodies indicated on the left.