ChIP Anti-bodies H3K4me3

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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Protocol tips
Immunoprecipitation was further performed with 150-μL sheared chromatin, 2-μg antibody, 50-μL Protein G sepharose beads and 800-μL dilution buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS) overnight at 4 °C. Next day, immuno-complexes were washed once with Wash buffer I (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), once with Wash buffer II (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), once with Wash buffer III (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% Na-deoxycholate, 1% NP-40), and twice with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The immune complexes were eluted twice with 100-μL elution buffer (1% SDS, 0 .1M NaHCO3, 20 mg/mL Proteinase K) at room temperature. The elution was incubated at 65 °C for 6 h and then purified with DNA purification kit (TIANGEN DP214-03).
Upstream tips
-Add protease inhibitors to all lysis solutions before use.
Protocol tips
-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.
-Do not place ethidium bromide in the agarose gel or the electrophoresis buffer, because of the presence of SDS.
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.
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