Immunohistochemistry γH2AX

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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