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

ChIP was performed using an EpiTect ChIP OneDay Kit (Qiagen, 334,471) and Simple ChIP Plus Sonication Chromatin IP Kit (Cell Signaling Technology, 56383S) following the manufacturer’s instructions. Briefly, cross-linking was performed using a 1%-formaldehyde solution in phosphate-buffered saline. Prior to immunoprecipitation, each input fraction was saved and used as a positive control. The supernatants were immunoprecipitated with anti-LRP1 carboxyterminal end, anti-CAPZA1, and anti-histone H3 (acetyl K9) antibodies or IgG from rabbit serum at 4°C overnight. Then, the resulting enriched genomic DNA samples were measured by qPCR using the EpiTect ChIP-qPCR Primer Assay kit for LAMP1 (Qiagen, GPH1003759(−)01A; −1 kb from the transcription start site), for CAPZA1 (Qiagen, GPH100073(-)01A; −1 kb from the transcription start site). These ChIP qPCR primers were pre-designed, and qPCR assays were optimized to measure genomic DNA in the region of −1 kb from the transcription start site of LAMP1 or CAPZA1.

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Manufacturer protocol

Download the product protocol from Qiagen for EpiTect ChIP OneDay Kit below.

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