%0 Journal Article
%T Dual agarose magnetic (DAM) ChIP
%A Lata Balakrishnan
%A Barry Milavetz
%J BMC Research Notes
%D 2009
%I BioMed Central
%R 10.1186/1756-0500-2-250
%X Protein A agarose and protein G magnetic beads bound with different IgGs have been combined in a single Chromatin Immunoprecipitation (ChIP) assay to analyze for the presence of two epitopes on the same chromatin at the same time. This procedure has been used with non-immune rabbit IgG bound to either the agarose or beads in order to include an internal negative control for non-specific binding of chromatin. The procedure has also been used with various antibodies including those targeting RNA Polymerase II and replication protein A 70 to determine whether specific forms of modified histones are present in either transcribing or replicating forms of SV40 minichromosomes respectively.The DAM ChIP procedure is a rapid, simple, and sensitive technique to characterize two epitopes located in the same chromatin. It should be particularly useful for the rapid screening of epigenetic modifications present in biologically active chromatin.Chromatin Immunoprecipitation (ChIP) has become an extremely powerful tool for the characterization of biologically functional chromatin. Specific antibodies bound to either protein A agarose or protein G magnetic beads are used to immune-select fragments of chromatin containing the target epitope of the antibody followed by PCR amplification to identify and quantitate the DNA present in the chromatin [1-4]. A carrier like agarose or magnetic beads is required in both of these procedures because it is necessary to separate the chromatin bound to antibody from contaminating chromatin during purifications. In the standard procedure this is done by sedimenting the agarose by low speed centrifugation, while in the modification with magnetic beads a magnet is used to bind the beads.We have taken advantage of the distinct properties of agarose and magnetic beads and developed a procedure which allows chromatin immunoprecipitation with two antibodies present in the same tube. A schematic representation of this procedure is shown in Figure 1. One
%U http://www.biomedcentral.com/1756-0500/2/250