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EP300 Antibody (730044) in ChIP
Chromatin immunoprecipitation analysis of p300 in mouse kidney was performed using biotinylated p300 antibody (Product # 730044). Kidney preparation: CD1 mice (Charles River Laboratories, Wilmington, MA; 6-10 weeks of age, male; 30-35 gms), received a tail vein injection of either LPS (2 mg/kg; 0111:B4; in 80 ?l of PBS) or PBS. Two hours after injection, mice were anesthetized, the abdominal cavity was opened, the kidneys were extracted, and renal cortical samples were cut from the kidneys with a razor blade and rapidly frozen at -70oC. The mice died within 30 sec due to exsanguinations. Chromatin preparation and multiplex Matrix ChIP platform: The multiplex microplate Matrix ChIP method was previously described (see reference for Matrix ChIP protocol: (http://www.ncbi.nlm.nih.gov/pubmed/22098709 ). Briefly, kidney fragments (10-20 mg) were cross-linked with formaldehyde, and chromatin was sheared. ChIP assays were done using either protein A- or streptavidin-coated 96-well polypropylene microplates. p300-biotin antibody (Product # 730044) was used at 0.5 ug/well. For Pol II antibody (0.5ug/well) testing, wells were pre-incubated with 1ug/well of goat anti-mouse biotinylated antibody (Product # B2763). 1ul of eluted DNA was used in 2 ul real-time PCR reactions (ABI7900HT) reactions containing primers shown to amplify exon 1 and exon 7 of the ICAM1 gene. PCR calibration curves were generated for each primer pair from a dilution series of total mouse or human genomic DNA. T
