Finding the right antibody for your ChIP experiment is essential. Not all antibodies work for all applications and you need to ensure your antibody works for ChIP and is specific for your target of interest. Some things to consider:
If the antibody has been validated for ChIP, then you can use the manufacturer’s guidelines and any published references to guide your experiment. However, in some cases your target of interest has not been tested in ChIP.
If the antibody has been validated in immunoprecipitation (IP), it has a high probability of working in ChIP. Other methods that require the antibody to recognize the target in a native state are also good indicators, such as immunofluorescence (IF), immunocytochemistry (ICC), and immunohistochemistry (IHC).
If the antibody has not been validated in ChIP, an excellent indicator is to perform a ChIP-western. Not only does a ChIP-western provide confidence that the antibody pulls down the protein of interest, it can also provide information about specificity. Although, a ChIP-western is labor intensive as the workflow is the same as ChIP through IP step, ChIP-westerns can be performed in parallel to ChIP using a fraction of the IP for ChIP-western and saving the rest for ChIP. After pulling down the protein, instead of eluting the DNA, the proteins are eluted from the beads by boiling and a western blot is performed. Probing the blot with a different antibody to the same target demonstrates that the antibody you chose is recognizing and immunoprecipitating an epitope of the protein of interest. Further, if you have blocking peptides available, specificity can be confirmed as the IP should not work or a significant decrease in efficiency should occur in the presence of the peptide.
Specificity of the antibody is a growing and understandable concern, particularly in ChIP-seq. Invitrogen antibodies undergo a two-part testing approach: functional application validation and targeted specificity verification. Functional application validation provides information on whether the antibody works in ChIP. Target specificity verification ensures the antibody is recognizing the target protein of interest. This can be achieved by several methods including the use of knockout and knockdown cell lines, treatment of cells that alters target expression levels, relative expression of the target of interest (if it is differentially expressed), and immunoprecipitation-mass spectrometry (IP-MS). Invitrogen antibodies that have been verified for specificity by one of these techniques have an Advanced Verification badge on our website.