HighCell# ChIP kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
After cell harvesting, chromatin must be sheared to 200 to 600 bp before ChIP. -Optimize shearing conditions for your specific cell type and fixation protocol before starting a ChIP. Therefore, start with a small sample (1x 10e5 to 1x 10e6 cells) and check the shearing efficiency.	-Keep the beads homogenously in suspension at all times when pipetting. Variation in the amount of beads will lead to lower reproducibility. -Make sure that there are no crystals in the Buffer B. Gently heat and mix until crystals disappear. -Chromatin from 1,000 cells is sufficient for one ChIP assay.	-For longer term storage, aliquot 130 µl of sheared chromatin into cryotubes, snap-freeze in liquid nitrogen, and store at -80°C.

Upstream tips
After cell harvesting, chromatin must be sheared to 200 to 600 bp before ChIP. -Optimize shearing conditions for your specific cell type and fixation protocol before starting a ChIP. Therefore, start with a small sample (1x 10e5 to 1x 10e6 cells) and check the shearing efficiency.
Protocol tips
-Keep the beads homogenously in suspension at all times when pipetting. Variation in the amount of beads will lead to lower reproducibility. -Make sure that there are no crystals in the Buffer B. Gently heat and mix until crystals disappear. -Chromatin from 1,000 cells is sufficient for one ChIP assay.
Downstream tips
-For longer term storage, aliquot 130 µl of sheared chromatin into cryotubes, snap-freeze in liquid nitrogen, and store at -80°C.

Publication protocol

Cells grown in 15 cm dishes in complete 6H medium for 48 hours (after initial plating of four million cells) were treated with 5 μM of SLF or 0.1% dimethyl sulfoxide vehicle. Following 24 hours of treatment, cells were used for chromatin immunoprecipitation (ChIP) assays using the HighCell ChIP kit (Diagenode, Liege, Belgium) following the manufacturer's instructions. A 1% portion of sheared cross-linked chromatin from each experimental condition was saved to serve as input DNA, and the rest was divided into equal parts and immunoprecipitated overnight at 4°C using 4 μg of rabbit-anti-Nrf2 polyclonal antibody (sc-13032 X; Santa Cruz Biotechnology) or 4 μg of rabbit IgG provided in the kit. Immunoprecipitated DNA were quantified by real-time RT-PCR using a standard curve from serial dilutions of input DNA and the primer pairs listed in Supplementary Table S7: (i) flanking the ARE1 sequence in the rat Tg promoter; (ii) flanking ARE2; (iii) flanking a validated ARE (positive control) in the rat Nqo1 promoter (34); or (iv) targeting an unrelated sequence (negative control) about 350 bp downstream of the 5′ end of the Actb gene. Quantified DNA for each target was normalized to corresponding input DNA, and the results were plotted as fold enrichment of the target sequence versus the negative control.

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Manufacturer protocol

Download the product protocol from Diagenode for HighCell# ChIP kit below.

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