Figure 2

DAM ChIPs. 48 hour unfixed SV40 minichromosomes were analyzed by (a) standard (Std ChIP) or (b, c, d) DAM chromatin immunoprecipitation (DAM ChIP) and the immunoprecipitates were amplified by PCR (32 cycles) with primer sets to the early region of the SV40 genome as previously described [5–7]. (a) Std ChIPs with antibodies bound to protein A agarose or protein G magnetic beads. Lane 1, Std ChIP with IgG (7.5 μl) (Bethyl Laboratories; P120-101) bound to agarose (Millipore, 17-295); lane 2, Std ChIP with Hyp H4 (7.5 μl) (Millipore; 06-866) bound to agarose; lane 3, Std ChIP with IgG (7.5 μl) bound to magnetic beads (Active Motif, 53014); lane 4, Std ChIP with Hyp H4 (7.5 μl) bound to magnetic beads. Ag; Agarose, MB; magnetic beads. (b) DAM ChIPs in which chromatin was analyzed with IgG (7.5 μl) bound to agarose (Lane 1) and Hyp H4 (7.5 μl) bound to magnetic beads (Lane 2) in the same tube or IgG (7.5 μl) bound to magnetic beads (Lane 3) and Hyp H4 (7.5 μl) bound to agarose (Lane 4) in the same tube. (c) DAM ChIPs immune selected with antibody to Hyp H4 bound to magnetic beads. Purified immune selected chromatin was divided into two aliquots and an aliquot was added to a tube containing either IgG or Hyp H3 bound to agarose. Lane 1, DAM ChIP with IgG (7.5 μl); lane 2, DAM ChIP with Hyp H3 (7.5 μl) (Millipore; 06-599). DAM ChIPs immune selected with antibody to Hyp H4 bound to agarose. Purified immune selected chromatin was divided into two aliquots and an aliquot was added to a tube containing either IgG or Hyp H3 bound to magnetic beads. Lane 1, DAM ChIP with IgG (7.5 μl); lane 2, DAM ChIP with Hyp H3 (7.5 μl).