Assessment of anti-Elf5 antibody for ChIP. The Elf5 antibody was tested for ChIP using the human cell line HEK293 UAS-TK-Luc, which contains a stably integrated luciferase reporter gene under the control of UAS (containing Gal4 binding sites) and the TK promoter. A. Schematic representation of the plasmids and primers utilized in the experiment. B. Expression of the Gal4 proteins assessed by anti-Gal4 and anti-Elf5 antibodies. HEK293 UAS-TK-Luc cells were transfected with plasmids that express Gal4 DNA-binding domain (Gal4 DBD) or Gal4 DBD-Elf5. C. ChIP assays performed with anti-Gal4 DBD or with anti-Elf5 antibodies. Immunoprecipitated DNA was analyzed using P1 and P2 primers. As a positive control, an aliquot (1%) of chromatin complex before immune isolation was used as input for PCR. Nonspecific binding was judged using rabbit IgG or no antibody. D. Schematic overview of the ChIP protocol used for the cloning of Elf5-putative target genes.
Escamilla-Hernandez et al. BMC Molecular Biology 2010 11:68 doi:10.1186/1471-2199-11-68