ChIP Anti-bodies H3K27me2

In ChIP, the most vital step is the binding of an antibody and choosing the right antibody. The binding affinity of different types of immunoglobulins to protein A or G differs significantly. Henceforth, it is recommended to choose either protein A or protein G coated beads. If you do not see any product in the positive control, add 5–10 μg of chromatin and 1–5 μg of antibody to each IP reaction and incubate with antibody overnight and an additional 2 hr after adding Protein G/A beads. If no product is observed in the experimental sample, add more DNA to the PCR reaction or increase the number of amplification cycles. Furthermore, if you have any problem with antibodies, make sure to use the ChIP-validated antibody.

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Found 3 matching solutions for this experiment

Protocol tips
ChIP assays were performed using the EpiQuik™ Tissue Chromatin Immunoprecipitation kit (Epigentek Group Inc.), as previously reported [13-15]. Briefly, small pieces of normal and PCOS ovarian tissues (1-2 mm3) were crosslinked with 1% formaldehyde. Cross-linking was terminated using 1.25 M glycine. The tissues and cells were added to homogenizing buffer, triturated, disaggregated, and centrifuged at 1000 g for 5 min at 4°C. After the removal of supernatants, protease inhibitors were added and the disaggregated tissue pellet was resuspended. Chromatin was sheared by sonication. Immunoprecipitation was performed at room temperature for 90 min.
Upstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.
Protocol tips
-For optimal ChIP results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP.
-It is important to keep the tissue cold to avoid protein degradation.
-Use fresh formaldehyde that is not past the manufacturer's expiration date.
Downstream tips
-Once in solution, store 1M DTT at -20°C.
Upstream tips
-Add protease inhibitors to all lysis solutions before use.
Protocol tips
-Use 2-25 µg for µg of chromatin.
-Keep cells on ice between the rounds of homogenisations.
- Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line.
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