EpiTect ChIP qPCR Assays

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) assay (EpiTect ChIP qPCR Assay kit; Qiagen) was performed to evaluate the extent of NF-κB binding to the DNA elements in the IL-6 promoter regions respectively using “EpiTect ChIP qPCR Assays kit” from Qiagen. ChIP assays were done as previously described [33]. Ipsilateral lesion side cortical tissue was exposed to 1% formaldehyde to cross-link with DNA proteins. We added glycine to each sample tube to quench unreacted formaldehyde. Cortical tissue was broken open with a sodium dodecyl sulfate (SDS) lysis buffer containing Protease Inhibitor Cocktail II (Calbiochem: Merck Millipore, Darmstadt, Germany). We sheared the chromatin to a manageable size by using a sonicator. Generally, between 200 and 1000 bp of DNA is used to achieve a high degree of resolution during the detection step. We added Protein G Agarose (Millipore, Billerica, MA) to each IP tube to remove proteins or DNA. We used an anti-NF-κB Ab (Millipore) or rabbit IgG (Millipore) to immunoprecipitate the precleared chromatin. It was then incubated overnight at 4 °C while being rotated. Protein G Agarose was added to each IP tube and incubated for 1 h at 4 °C with rotation to collect the antibody/antigen/DNA complex. After four consecutive washes using four different wash buffers, the DNA from the DNA-protein complexes from all the samples, including the input and negative control, was reverse cross-linked by incubation with 2 μL of Proteinase K for 2 h at 65 °C. DNA was purified to remove chromatin proteins and to prepare it for the detection step. Primers specific for IL-6 promoters were used to determine the extents of both immunoprecipitated DNA and quantitative PCR. The following IL-6 primers were used: forward, 5′-GCG ATG GAG TCA GAG GAA AC-3′, and reverse 5′-TGA GGC TAG CGC TAA GAA GC-3′. Results are presented as a percentage of input.

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Manufacturer protocol

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