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

Raw 264.7 macrophage cells purchased from ATCC were cultured in standard DMEM containing 10%FBS and penicillin/streptomycin at 37 degrees in 5% CO2. Cells were transfected using Lipofectamine 2000 according to the manufacturer’s protocol. For ChIP-seq experiments, mouse Zhx2 was cloned into a pShuttle-IRES-GFP1 plasmid (ad-gene) where the C-terminal end of the gene contained a 3X-FLAG sequence.

Transfected Raw macrophages were washed 2X in cold PBS, fixed using 4% paraformaldehyde, neutralized with acid glycine then pelleted at 2million cells/pellet. These pellets were then fractionated for nuclear isolation using an NPER kit (Thermo cat# 78833). Nuclear lysate was then sonicated to ~150–300 DNA base-pair fragment size for 50min using a Bioruptor ultrasonicator (diagenode). Sonicated lysate was then subjected to ChIP using a Kit (Sigma cat #CHP-1) according to manufacturer details. Briefly, sonicated pellets were immuoprecipitated using 3ug/2million cells monoclonal FLAG antibody (Sigma cat# F3165) overnight at 4 degrees. The following day, samples were washed and cross-links reversed via incubation overnight at 65 degrees in 160 mM NaCl. Protein and RNA was then digested (Proteinase K and RNAse digestion 1hr at 37 degrees), and isolated DNA eluted using a column provided in ChIP-kit.

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Manufacturer protocol

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