ChIP Troubleshooting Tips

Problems Suggested Solutions
High background in negative control (IgG or mock IP) samples Excessive antibody resulting in binding to non-targets: Optimize the concentration of the antibody.
Nonspecific binding to beads: Include a pre-clearing step to exclude these non-targets or add a blocking agent to the beads.
Incomplete fragmentation of chromatin: Optimize the fragmentation process to acquire chromatin lengths between 200-1000 bp. Separately optimize fragmentation for each cell or tissue type. Use siliconized or low retention tubes.
Contaminated reagents: Ensure that all reagents are freshly prepared and free of contaminants. Increase the number of washes.
Run a “no DNA” PCR reaction to determine if your sample is contaminated with nucleic acids.
“No DNA” PCR reaction is showing signal Use pipettes dedicated to PCR, and UV-irradiate pipettes prior to setting up PCR.
Perform ChIP, DNA purification, and PCR reaction setup in three separate rooms/areas using dedicated pipettes, or set up reactions in a hood.
Avoid using bottled or otherwise prepackaged water. Use freshly delivered Milli-Q® water from a system containing a UV light source.
Use aerosol-resistant pipette tips.
Do not open tubes containing amplified PCR products anywhere near the location of future ChIP or qPCR experiments.
No amplification with input DNA Your input DNA sample should be taken just prior to adding the antibody. It is considered the starting material. If you are not seeing amplification with your input DNA, either you have not successfully reversed the cross links or the PCR is not working for reasons other than the kit.
low recovery of DNA Ineffective or low affinity ChIP antibody: Ensure that you are using an antibody that has been validated in ChIP For a complete selection of ChIP antibodies. If you are using a ChIP antibody, increase the incubation time of the antibody.
Insufficient ChIP antibody: Generally, 1-10 µg of ChIP antibody is sufficient. However, the amount of antibody required may depend on the relative abundance of your target protein and the affinity of the antibody for the target.
Insufficient starting sample: Before crosslinking, prepare a separate plate to determine cell number. Re-evaluate your cell number per reaction. Increase your cell number especially if you are attempting to detect a low abundance target.
Incomplete cell lysis and ineffective fragmentation: Optimize these steps by varying parameters  and assessing their effects on chromatin recovery. Use mechanical force such as a dounce homogenizer or glass beads to improve cell lysis. Optimize the fragmentation steps and avoid foaming.
Over crosslinking: Long incubation in formaldehyde may mask epitopes required for recognition by ChIP antibody. This can be especially problematic if you are using a monoclonal antibody. Over crosslinking may also result in the formation of complexes that are resistant to sonication. Optimize crosslinking steps: the final concentration of formaldehyde should be 1%, and you should determine the most effective crosslinking time before proceeding with the experiment.
Under crosslinking: Insufficient crosslinking may result in dissociation of target proteins from DNA during subsequent steps of the protocol. Unless you are studying histones and histone modifications, you should use an X-ChIP protocol to stabilize the associations of your target protein with DNA. Increase crosslinking time.
low affinity or low quality beads: Protein G magnetic beads bind a wider range of antibodies, including mouse monoclonals. For the most flexibility with antibody choice, we recommend the protein A/G blend (Cat. #: 16-663).
PCR primers: Test the efficiency of the primers. Include appropriate controls and redesign primers if necessary.
Pull down only large DNAs Over-fixation and heating during fixation and sonication can inhibit fragmentation: A long sonication time may damage the epitope. Shortening your fixation times will allow you to shorten your sonication times and get smaller fragments. Also, we recommend to run a gel of the pull-down DNA before IP to see if a blazing band of fragmented DNA appears at ~200-300bp and nothing else on the gel, to make sure uniform fragmentation is achieved.
Fixation is not quenched by glycine
more DNA is precipitated in my no-antibody control than for my test sample
To eliminate banding in your negative controls you can do several things:

A) Pre-clear the 2ml diluted cell pellet suspension with 80 microliters of Salmon Sperm DNA/Protein A Agarose-50% Slurry (Cat. #: 16-157) for 30 minutes at 4ºC with agitation. You could try to preclear the lysate longer or with more clearings.

B) Titrate your input DNA, to see when the bands in the NFA disappear.

C) Use an alternative lysis procedure: Resuspend cell pellet in 200 microliters of 5mM Pipes pH 8.0, 85mM KCl, 0.5% NP40 containing protease inhibitors. Place on ice for 10 minutes. Pellet by centrifugation (5 minutes at 5000 rpm). Resuspend pellet in 200 microliters of 1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1 containing protease inhibitors. Incubate on ice for 10 minutes.

D) Block the Salmon Sperm DNA Agarose prior to use in 1-5% BSA and Chip dilution buffer (mix at room temperature for 30 minutes). After incubation, spin the agarose and remove the 1% BSA/ChIP assay buffer supernatant. Wash once in ChIP assay buffer and continue.

 

For more information about ChIP experiment guides, troubleshooting tips and supplementary protocols, please view our Chromatin Immunoprecipitation (ChIP) page.