What are the factors to consider when planning ChIP assays?
Posted July 28, 2023
When planning ChIP assays, important factors to consider include: chromatin quality, antibody validation, cross-linking issues, cell number considerations, and appropriate controls. Sonication is typically used to break down chromatin into shorter fragments. Important variables to consider when breaking down chromatin are cell density and sonication power, time, temperature, and the number of cycles. For antibody recognition, ChIP requires recognition of tertiary structure of the target protein and of an exposed epitope. It is recommended to use ChIP grade and ChIP-validated antibodies. However, antibodies validated for IHC or IP may be used as well. If there is no suitable antibody, it is also possible to tag a target protein with a his, GST, or myc tag. To minimize cross-linking issues, cross-linking should only be performed for a short period of time, no longer than 10 minutes. Excessive cross-linking reduces antigen availability and efficiency of the chromatin-shearing step. It may also mask the epitope that binds to the antibody, leading to a reduction in protein that can be pulled down. It is also important to optimize the amount of cells in the experiment because it is dependent on the abundance of the target protein being studied. Lastly, the experiment should have the appropriate controls such as IgG control, positive control, negative control, and input control.