Anti-Histone H3 (acetyl K9) antibody - ChIP Grade (ab4441)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Add protease inhibitors to all lysis solutions before use.	-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. -Do not place ethidium bromide in the agarose gel or the electrophoresis buffer, because of the presence of SDS.

Upstream tips
-Add protease inhibitors to all lysis solutions before use.
Protocol tips
-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. -Do not place ethidium bromide in the agarose gel or the electrophoresis buffer, because of the presence of SDS.

Publication protocol

Chromatin immunoprecipitation (ChIP) was performed according to the manufacturer’s instruction (CST). Approximately 4 × 106 cells were prepared for each immunoprecipitation (IP). Briefly, TZM-bl cells were treated with siNC, siTRIM28 or TNFα (PeproTech) for 48 hr followed by crosslinking proteins to DNA with 1% formaldehyde (Sigma-Aldrich) for 10 min at room temperature. The fixation was quenched with 125 mM glycine for 5 min at room temperature followed by centrifuging at 1,500 rpm for 5 min at 4°C. The supernatants were removed immediately. Cell pellets were resuspended in ice-cold Buffer A (CST) supplemented with DTT and Protease inhibitor cocktail (PIC) and incubated on ice for 10 min. The nuclei were enriched by centrifugation at 3000 rpm for 5 min at 4°C and resuspended in ice-cold Buffer B (CST) supplemented with DTT. Nuclei pellets were centrifuged again, removed supernatants and resuspended in 100 μl Buffer B supplemented with DTT and 0.5 μl micrococcal nuclease (CST) per IP preparation. The digestion was conducted at 37°C for 20 min. Incubation tubes were inverted several times per 5 min. After digestion, the reaction was stopped by adding 50 mM EDTA followed by centrifugation at 13,000 rpm for 1 min at 4°C. Nuclei pellet was resuspended in 100 μl ChIP Buffer (CST) supplemented with PIC per IP preparation and incubated for 10 min on ice. The nuclei pellet was further lysed by sonication with 3 sets of 20 s pulses at 40% amplitude. Pellet was incubated on ice for 30 s between pulses. The lysates were clarified by centrifugation at 10,000 rpm for 10 min at 4°C. The supernatants which contained digested chromatin were transferred into new tube. One-tenth of the chromatin sample was proceeded to analyze the size and concentration. Briefly, 50 μl chromatin sample was removed RNA by RNase A (CST) and reversed cross-linking by 200 mM NaCl and Proteinase K (CST). DNA from samples were purified by DNA purification spin columns (CST). Concentration was determined by measuring OD260. The size range was analyzed by electrophoresis on a 1% agarose gel, which should be between 150 and 900 bp.

For each IP preparation, approximately 10 μg chromatin was diluted into ChIP Buffer. Ten microliter diluted chromatin, which was 2% input sample, was transferred to a new tube and stored at −20°C. Immunoprecipitation antibodies normal rabbit IgG (CST, 2729), anti-TRIM28 antibody (Proteintech, 15202–1-AP), anti-H3K9me2 antibody (Abcam, ab1220), anti-H3K9me3 antibody (Abcam, ab8898), anti-H3K4me3 antibody (Abcam, ab8580), anti-H3K27me3 antibody (Abcam, ab6002), anti-H3K9Acetyl antibody (Abcam, ab4441), anti-CDK9 antibody (CST, 2316), and anti-RNA polymerase II CTD repeat YSPTSPS (phospho Ser2) antibody (Abcam, ab5095) were separately added to siNC and siTRIM28 groups, respectively. The immunoprecipitation was carried out overnight at 4°C while rotating. ChIP-Grade Protein G Magnetic Beads (CST) were added to the each IP reaction and incubated with IP samples for another 2 hr at 4°Cwhile rotating. The protein G magnetic beads were pelleted by placing the IP tubes in a magnetic separation rack and washed with 3 times low-salt washes and one time high-salt wash. Each wash was conducted at 4°C for 5 min while rotating. DNA enriched by protein G magnetic beads was eluted by ChIP Elution Buffer (CST). All the DNA samples including 2% input samples were reversed cross-linking with 200 mM NaCl and Proteinase K and purified as above.


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Manufacturer protocol

Download the product protocol from Abcam for Anti-Histone H3 (acetyl K9) antibody - ChIP Grade (ab4441) below.

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