EpiTect ChIP OneDay Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Warm IP Lysis Buffer to room temperature to prevent precipitation. -Prepare one extra plate of cells to estimate cell number. -Thaw Protease Inhibitor Cocktail (PIC) at room temperature. -It is recommended to take some trial to optimize sonication condition before beginning with sonication.	- In general it is recommended that one million mammalian cells are required for each IP fraction. -Centrifuge the sample at 6000 × g for 5 min at 4 °C to eliminate bubbles. -To reduce background, remove as much supernatant as possible in the last wash. Continue with next step OR store at 4°C overnight.

Upstream tips
-Warm IP Lysis Buffer to room temperature to prevent precipitation. -Prepare one extra plate of cells to estimate cell number. -Thaw Protease Inhibitor Cocktail (PIC) at room temperature. -It is recommended to take some trial to optimize sonication condition before beginning with sonication.
Protocol tips
- In general it is recommended that one million mammalian cells are required for each IP fraction. -Centrifuge the sample at 6000 × g for 5 min at 4 °C to eliminate bubbles. -To reduce background, remove as much supernatant as possible in the last wash. Continue with next step OR store at 4°C overnight.

Publication protocol

Chromatin immunoprecipitation (ChIP) was done using EpiTect ChIP OneDay Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. MIN6 cells were cultured in T25 flasks until 80% confluency. The proteins were cross-linked to DNA using 1% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 10 minutes. The cells were then harvested and lysed using Lysis Buffer containing protease inhibitor cocktail and sonicated with Bioruptor® sonication device (Diagenode, Liege, Belgium) for the generation of chromatin fragments. The lysate was pelleted and supernatant pre-cleared with Protein A beads. Specific antibodies have to be used for the immunoprecipitation of chromatin fragments to which the protein of interest is bound. For this, precleared lysate was separated into three IP fractions, and incubated at 4°C overnight with mouse control IgG (Genei, Bangalore, India) as negative control, anti-RNA polymerase II as positive control or anti-STAT5B for the protein of interest. 1% of IP fraction was kept as input fraction. Since RNA polymerase II is present at the promoters of all actively transcribed genes, it was used as the positive control. The IP fractions containing protein of interest bound to the corresponding chromatin region were immunoprecipitated with Protein A beads. Genomic DNA was isolated and purified, followed by RT-PCR using EpiTect ChIP qPCR Primer Assay for mouse Ccnd2, a known STAT5B target, and Nos2, the region under investigation (Qiagen, Hilden, Germany).

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Manufacturer protocol

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