Chromatin immunoprecipitation (ChIP) assays identify DNA-protein interactions in the genome to understand transcription regulation through histone modifications, transcription factors, and the chromatin occupancy of chromatin remodelers. The strength of ChIP assays is the ability to capture a snapshot of specific DNA-protein interactions occurring in the cell and to quantitate the interactions using quantitative polymerase chain reaction (qPCR) or ChIP-sequencing (ChIP-Seq). ChIP experiments require a variety of proteomics and molecular biology methods including crosslinking, cell lysis (DNA-protein extraction), nucleic acid shearing, antibody-based immunoprecipitation, DNA sample clean-up, and PCR.
Considerations when choosing a ChIP antibody
- Whether it will work in ChIP. Not all antibodies work for all applications. If the antibody has been validated* for ChIP, then the manufacturer’s guidelines or any published references can be used as guidance for the experiment. However, in some cases, if the target of interest has not been tested in ChIP but has been tested in immunoprecipitation (IP), there is a high probability the antibody will work 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). ChIP-western is another method that provides confidence the antibody pulls down the protein of interest and can provide information about specificity.
- Whether it is specific. Specificity of the antibody is a growing and understandable concern, particularly in ChIP. Invitrogen antibodies are undergoing a two-part testing approach: functional application validation* and targeted specificity verification. Functional application validation provides information on whether the antibody immunoprecipitates in ChIP. Target specificity verification ensures the antibody is recognizing the protein of interest.
Both monoclonal and polyclonal antibodies can work in ChIP. The key requirement is that the specific epitope of interest is exposed. One of the advantages of using a monoclonal antibody is that, generally, it is more specific. However, this is associated with a higher likelihood that the one epitope it recognizes is buried. Unless monoclonal antibodies are specifically screened or designed for use in ChIP, polyclonal antibodies are better candidates for recognizing target proteins, as they recognize multiple epitopes of the targets. Invitrogen also offers recombinant polyclonal antibodies, which have the advantage of recognizing multiple epitopes like a traditional polyclonal antibody but is recombinant, ensuring lot-to-lot consistency. An alternative approach, if the target of interest does not have an antibody that works for ChIP, is to tag the target with Myc, His, human influenza hemagglutinin (HA), T7, GST, or V5.
ChIP data examples
Each Invitrogen antibody that is indicated for ChIP applications has undergone functional application testing. Here are some examples of that testing.
Chromatin Immunoprecipitation (ChIP) assay of endogenous HAT1 protein using Anti-HAT1 Antibody. ChIP was performed using HAT1 Recombinant Rabbit polyclonal Antibody (Cat. No. 712581, 5 µg) on sheared chromatin from 2 million HeLa cells. Normal rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to transcriptional start site and gene body (+2 Kb) of GAPDH, RPL30 transcriptional start site, CDKN1A intron 1, and SAT2 satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.
Chromatin Immunoprecipitation (ChIP) assay of Histone H3K4me1 protein using Anti-Histone H3K4me1 Antibody. ChIP was performed using Histone H3K4me1 Recombinant Rabbit Polyclonal Antibody (Cat. No. 710795, 5 µg) on sheared chromatin from 2 million Jurkat cells. Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR with optimized PCR primer pairs for the promoters of the active cFOS, beta-actin genes, and IRF1 Exon 2 region used as positive control target gene, and the region of the inactive SAT2 satellite repeat, used as negative control target gene. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.
*The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.
For Research Use Only. Not for use in diagnostic procedures.