Recombinant Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Add protease inhibitors to all lysis solutions before use.	-Use 4 µg for 25 µg of chromatin. -Keep cells on ice between the rounds of homogenisations. - Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line.

Upstream tips
-Add protease inhibitors to all lysis solutions before use.
Protocol tips
-Use 4 µg for 25 µg of chromatin. -Keep cells on ice between the rounds of homogenisations. - Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line.

Publication protocol

ChIP experiments were performed using the EZ ChIP Chromatin Immunoprecipitation Kit (Millipore, Billerica, USA). Four primer sets were designed to flank-related putative ERα-binding sites in the promoter region of vinculin. Details of the primer sequence are listed in Supplementary Table 9. Briefly, MCF-7 cells were fixed with 1% paraformaldehyde and sonicated seven times for 10 s each using a sonicator with a microtip in a 1.5-ml tube. Anti-ERα antibody or control human IgG was applied to pull down the chromatin associated with ERα. The chromatin–antibody complexes were collected with Protein G-Agarose. After washing and elution of the complexes from the beads, the DNA–protein crosslinks were reversed at 65 °C overnight. The amounts of the specific DNA fragment were then quantified by real-time PCR and normalized against the genomic DNA preparation from the same cells. Each group was made in triplicate.

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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063) below.

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