EpiQuik Acetyl-Histone H4 ChIP Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Always cap spin columns before placing them in the microcentrifuge. -Ensure that all buffers are in clear solution. Shake or vortex if these buffers precipitate. -The conditions of cross-linked DNA shearing can be optimized based on cells and sonicator equipment. If desired, remove 5 µl of sonicated cell lysate for agarose gel analysis.

Protocol tips
-Always cap spin columns before placing them in the microcentrifuge. -Ensure that all buffers are in clear solution. Shake or vortex if these buffers precipitate. -The conditions of cross-linked DNA shearing can be optimized based on cells and sonicator equipment. If desired, remove 5 µl of sonicated cell lysate for agarose gel analysis.

Publication protocol

cccDNA ChIP assays were performed using EpiQuik Acetyl-Histone H3 ChIP and EpiQuik Acetyl-Histone H4 ChIP kits (EpiGentek, Farmingdale, NY, United States). Briefly, cells were collected and in vivo cross-linked in fresh culture medium containing 1% formaldehyde for 10 min at RT and were then lysed in 200 μL CP3A for 10 min at RT to isolate nuclear pellets. Chromatin solutions were sonicated for 4 pulses of 12 s each at level 5 using a Branson Microtip probe, followed by a 40-s rest on ice between each pulse to generate 200- to 1000-bp DNA fragments. Supernatants were diluted with CP4 at a 1:1 ratio, and 5 μL was removed as “input DNA”. Chromatin was then subjected to immunoprecipitation for 1 h at RT using strip wells pre-coated with antibodies specific to acetyl-histone H3, acetyl-histone H4 or normal mouse IgG. After six washes with CP1, immunoprecipitated chromatins and input DNA coated on the strip wells were digested with proteinase K and then purified using collection tubes. Purified DNA was subjected to Plasmid-Safe ATP-Dependent DNase digestion and real-time PCR amplification, as described above.

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Manufacturer protocol

Download the product protocol from Epicentre for EpiQuik Acetyl-Histone H4 ChIP Kit below.

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