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ChIP was performed on sheared chromatin from 4,000,000 HeLa cells. A titration consisting of 1, 2, 5 and 10 ug of antibody per ChIP experiment was analyzed. IgG (2 ug/IP) was used as a negative IP control. Quantitative PCR was performed with optimized primers for the H19 imprinting control region, and a specific region in the GAPDH gene, used as positive controls, and for the MYOD1 gene and the Sat2 satellite repeat region, used as a negative control. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

ChIP was performed on sheared chromatin from 4,000,000 HeLa cells using 1 ug antibody. The figure shows the peak distribution along the complete sequence and a 1 mb region of the X-chromosome and in two regions surrounding the H19 and GAPDH positive control genes, respectively.

Western Blot (Cell lysate) analysis of 25 ug whole cell extracts of HeLa cells.

Western Blot (Cell lysate) analysis of (1) Negative control, and (2) 40 ug whole cell extracts of HeLa cells transfected with CTCF siRNA.

Immunofluorescent staining of Hela cell line with antibody followed by an anti-rabbit antibody conjugated to Alexa488 (left). The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings (right).

ELISA is a quantitative method used to determine the titer of the antibody using a serial dilution of antibody against CTCF. The plates were coated with the peptides used for immunization of the rabbit. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be >1:500000.