Imprint® Chromatin Immunoprecipitation Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-If any reagent has formed a precipitate, warm at 55 °C until dissolved. Allow to equilibrate to room temperature before use.	-Use 1 µg of antibody per well. -The range of cells that can be used with this kit is 0.2–2.5 x 105/well/ChIP sample. Using more than 0.5 million cells per well will increase the non-specific binding and reduce the yield and specificity of the ChIP reaction. If higher yield of ChIP DNA is desired for downstream applications (e.g. ChIP-Seq., ChIP-chip): a) DNA could be pooled by eluting consecutively (with 50 µl of elution buffer) from multiple individual ChIP reactions.

Upstream tips
-If any reagent has formed a precipitate, warm at 55 °C until dissolved. Allow to equilibrate to room temperature before use.
Protocol tips
-Use 1 µg of antibody per well. -The range of cells that can be used with this kit is 0.2–2.5 x 105/well/ChIP sample. Using more than 0.5 million cells per well will increase the non-specific binding and reduce the yield and specificity of the ChIP reaction. If higher yield of ChIP DNA is desired for downstream applications (e.g. ChIP-Seq., ChIP-chip): a) DNA could be pooled by eluting consecutively (with 50 µl of elution buffer) from multiple individual ChIP reactions.

Publication protocol

ChIP was performed using the Imprint Chromatin Immunoprecipitation Kit (Sigma-Aldrich) according to manufacturer’s instructions. Briefly, microvessels were isolated from Nx, Hx, H/R, and sulforaphane treated rats and subsequently cross-linked in buffer containing 1% formaldehyde for 10 min at room temperature. Cross-linking was stopped by addition of glycine to a final concentration of 125 mM followed by centrifugation at 180×g for 5 min at room temperature. At this time, the microvessel pellet was resuspended in 50 μl of Nuclei Preparation Buffer and incubated on ice for 10 min. Following centrifugation at 180×g for 10 min at 4 °C, the nuclear pellet was resuspended in shearing buffer and incubated on ice for 10 min. Chromatin was sheared to 200–1000 bp by sonication on ice. Sonicated chromatin was diluted twofold in lysis buffer and 100 μl of diluted sample per immunoprecipitation reaction was used. Each sample was added to individual wells of a 96-well assay plate where each well contained 1 μg of specific rabbit monoclonal anti-Nrf2 antibody (EP1808Y) that has been previously validated in ChIP assays [21]. Assay plates were incubated for 90 min at room temperature on an orbital shaker at 75 rpm. In parallel, a no-antibody sample was run as a negative control. At this time, 40 μl of DNA release buffer was added to each well and samples were incubated in a water bath at 65 °C for 15 min. Following this step, 40 μl of reversing solution was added to each well and samples were incubated in a water bath at 65 °C for 90 min. Washes and elutions were performed in accordance with manufacturer’s instructions for the Imprint ChIP assay kit. Eluted and input DNA samples were purified using a spin column to a final volume of 50 μl. Quantitative real-time PCR was performed using 2 μl of template DNA per 25 μl of polymerase chain reaction (PCR) amplification scale as described by Hoque and colleagues [22]. Quantification of Nrf2 occupancy to the ARE within Abcc gene promoter by SYBR green real-time PCR was performed using primer sets prepared by Integrated DNA Technologies (Table 2). All measurements were performed in triplicate and results were verified in three separate chromatin preparations.

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for Imprint® Chromatin Immunoprecipitation Kit below.

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