ChIP Anti-bodies H3K4me1

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
Immunoprecipitations were carried out in 1 ml of IP buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 % Triton-X 100). Three µg antibody was used per 20 µg purified chromatin. Input chromatin was obtained after preclearing by de-crosslinking and purified using the Qiaquick column (Qiagen) according to manufacturer’s instructions. Immunoprecipitations were carried out with inverting at 4 °C for 14–16 h
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.
Upstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.
Protocol tips
-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.
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