Magna ChIP™ A - Chromatin Immunoprecipitation Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Make fresh formaldehyde before each experiment	--Just because an antibody works well in a Western blot does not always indicate it will perform well in chromatin immunoprecipitation. Unlike a Western blot that detects proteins that have been denatured, a ChIP antibody must recognize the target protein in its native state. Use ChIP-validated antibody. -Use 2-10 μg of your ChIP antibody depending on the abundance of your protein target.

Upstream tips
-Make fresh formaldehyde before each experiment
Protocol tips
--Just because an antibody works well in a Western blot does not always indicate it will perform well in chromatin immunoprecipitation. Unlike a Western blot that detects proteins that have been denatured, a ChIP antibody must recognize the target protein in its native state. Use ChIP-validated antibody. -Use 2-10 μg of your ChIP antibody depending on the abundance of your protein target.

Publication protocol

One-mL chromatin-protein aliquots were precleared with 100 μL 50% DNA/Protein A/G slurry (Salmon Sperm DNA/Protein G Agar, catalog # 16-201; SSDNA/Protein A Agar, catalog # 16-157; Normal Mouse IGG, catalog # 12-371, EMD-Millipore Corporation, Billerica, MA). Precleared lysates were incubated overnight at 4 °C with rotation with 4 μg of anti-H3AcK9 (Anti-Acetyl Histidine H3-lys 9, catalog # 06-942 from Millipore) or 4 μg of normal mouse IgG (Normal Mouse IgG, catalog #12-371). Immunocomplexes were transferred to 1.5-mL tubes containing 100 μL of Salmon Sperm DNA/Protein A/G Agarose-50% slurry and incubated with rotation at 4 °C for 4 h. Agarose-immunocomplexes were pelleted by centrifugation at 750 rpm for 1 min at 4 °C. The supernatant was carefully removed and the agarose-immunocomplexes were subjected to two washes with low-salt buffer (150 mM NaCl, pH 8.0), two washes with high-salt buffer (500 mM NaCl, pH 8.0) and two washes with LiCl buffer (250 mM LiCl, pH 8.0), followed by one wash with Tris-EDTA (TE).

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Manufacturer protocol

Download the product protocol from Merck Millipore for Magna ChIP™ A - Chromatin Immunoprecipitation Kit below.

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