ChIP Anti-bodies H3K36me3

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 in the experimental, add more DNA to the PCR reaction or increase the number of amplification cycles. Choose an alternate, ChIP-validated antibody if the antibody does not work.

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Found 3 matching solutions for this experiment

Upstream tips
-Add protease inhibitors to all lysis solutions before use.
Protocol tips
-Use 4µg for 106 cells.
-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.
Protocol tips
-5 - 10 µg per ChIP
-Avoid over fixation.
-purchase expensive, "hing end" sonicator to get reproducible shearing.
-Avoid repeated freeze/thaw cycles.
-Keep all reagents on ice when not in storage.
Protocol tips
Chromatin was extracted from naive CD4+Foxp3− T cells polarized for 48–72 h under various conditions (1×106 cells) after fixation with formaldehyde.
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