ChIP Anti-bodies H3K9-Ac

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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Protocol tips
The ChIP assay was performed using the imprint chromatin immunoprecipitation kit (CHP1, Sigma-Aldrich) following the manufacturer's instructions. The sonicated DNA was incubated with anti-IgG (Millipore, Milford, MA, USA) as a negative control. The purified DNA was amplified using specifically designed real-time ChIP PCR primers spanning the region −300 to +1000 of LY6K.
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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