HEF enrichment without protein–protein crosslinking and chromatin purification

Abstract

<p><b>Copyright information:</b></p><p>Taken from "A genomic approach to the identification and characterization of HOXA13 functional binding elements"</p><p>Nucleic Acids Research 2005;33(21):6782-6794.</p><p>Published online 30 Nov 2005</p><p>PMCID:PMC1301594.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () ChIP was performed in HOXA13-FLAG and HOX (−) cells using anti-FLAG agarose. Elimination of crosslinking with DMA preceding formaldehyde crosslinking is represented in indicated lanes [DMA(−)]. HEF1 and HEF2 are enriched upon addition of DMA as well as without DMA in the HOXA13-FLAG versus HOX (−) cells. HEF1 demonstrated a visibly higher signal with DMA versus DMA (−) in the HOXA13-FLAG cells (1.9-fold by BioRad Quantity One analysis) while HEF2 recovery was equal between the samples. () ChIP was performed in HOXA13-FLAG and HOX (−) cell lines using anti-FLAG agarose. CsCl purification of chromatin was eliminated in the indicated samples (−). HEF1 and HEF2 are both enriched in the HOXA13-FLAG cells versus the HOX (−) cells both with and without chromatin purification. There was consistently no product in the HOX (−) sample for HEF1; however, there is a product present in the CsCl purified HEF2 sample