Estrogen Receptor α (D8H8) Rabbit mAb #8644

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.	-For optimal ChIP and ChIP-seq results, use 5 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. -It is important to keep the tissue cold to avoid protein degradation. -Use fresh formaldehyde that is not past the manufacturer's expiration date.	-Once in solution, store 1M DTT at -20°C.

Upstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.
Protocol tips
-For optimal ChIP and ChIP-seq results, use 5 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. -It is important to keep the tissue cold to avoid protein degradation. -Use fresh formaldehyde that is not past the manufacturer's expiration date.
Downstream tips
-Once in solution, store 1M DTT at -20°C.

Publication protocol

ChIP assay was performed in our previous study. MCF‐7 cells were fixed for cross‐linking for 30 minutes. Subsequently, the cells were mixed with 0.1375 M glycine, washed with cold PBS/1 mmol L−1 PMSF and scratched into PBS/1 mmol L−1 PMSF for centrifugation. Cells were treated with SDS lysis buffer and sonicated for 10 minutes (30 seconds on/off). A ChIP assay kit (Millipore, 17‐295, Burlington, MA, USA) was used for the subsequent steps. The following antibodies were used in the ChIP experiments: anti‐STAT1 (cat. no. 9172) and anti‐ ERα rabbit (D8H8, cat. no. 8644).

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for Estrogen Receptor α (D8H8) Rabbit mAb #8644 below.

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