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

A ChIP assay was performed using an Imprint Chromatin Immunoprecipitation Kit (Sigma, St. Louis, MO, USA) according to the manufactures protocol. Briefly, MCF-7 cells were fixed with 1 % formaldehyde and quenched with 1.25 M glycine. After washing with PBS, the cells were suspended in nuclei preparation buffer and shearing buffer, and sonicated under optimized conditions. This sheared DNA was then centrifuged and a cleared supernatant was used for protein/DNA immunoprecipitation. The clarified supernatant was diluted with dilution buffer (1:1 ratio) and 5 μl of diluted samples were removed as an internal control. The diluted supernatant was incubated with antibody (STAT5b) in pre-coated wells for 90 min. For negative and positive control, normal mouse IgG and anti-RNA polymerase II were used, respectively. The unbound DNA was washed off with IP wash buffer and the bound DNA was collected by cross link reversal using DNA release buffer containing proteinase K. The released DNAs and the DNA from the internal controls were purified with GenElute Binding Column G. The DNA was then quantified using conventional PCR.

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Manufacturer protocol

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

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