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

Steroid receptor coactivator (SRC)‐1 recruitment to the HNF4α responsive elements (DR‐2) and E‐box in the OCT1 gene promoter was determined using a chromatin immunoprecipitation (ChIP) assay in the HepG2 cells, which were transfected with pSG5‐hPXR and pSG5‐FLAG hSRC‐1 (3.2 μg in 25 cm2 flask) expression plasmids after one day of cultivation. Cells were then treated using 10 μM rifampicin or 1‰ DMSO as control sample for 24 h. After the interval, the cells were harvested, and immunoprecipitation was conducted with the Imprint® Chromatin Immunoprecipitation Kit (Sigma‐Aldrich) according to the manufacturer's protocol. Anti‐FLAG M2 antibody (Sigma‐Aldrich), normal mouse IgG antibody and anti‐RNA polymerase II (Pol II) antibody were used for precipitation. Levels of SRC‐1 recruitment to the HNF4α response elements and into the E‐box were analysed by qRT‐PCR with primers and probes specifically designed to amplify indicated regions (Figure 6, upper panel, arrows). Data are presented as a relative binding to control vehicle (DMSO)‐treated cell samples (100%) immunoprecipitated with the same antibody. Non‐specific immunoprecipitation with mouse IgG antibody was 1% lower than with anti‐FLAG or the anti‐Pol II antibodies.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing ChIP Human - HepG2 using Imprint® Chromatin Immunoprecipitation Kit from Sigma-Aldrich.

Manufacturer protocol

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

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing ChIP Human - HepG2 using Imprint® Chromatin Immunoprecipitation Kit from Sigma-Aldrich. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.