| Target protein is not enriched at region of interest | Check available literature to determine if target was expected at binding locus Include positive and negative binding loci controls for target Include positive control antibody to confirm that ChIP is working as expected |
| Target protein interacts with DNA weakly or indirectly | Longer crosslinking may be required |
| N-ChIP protocol inappropriate for target | N-ChIP is suitable for histone proteins, which have an extremely strong interaction with DNA. It is not suitable for proteins with weak binding properties |
| Insufficient starting sample | Prepare a separate plate of cells to accurately determine cell number. Increase number of cells used if target is low abundance |
| Incomplete cell lysis | Try alternative buffers with a higher concentration of detergent, including separate cell and nuclear lysis buffers Check lysis progression under a microscope Include mechanical force in lysis step, such as a Dounce homogenizer |
| Degradation of sample occurred | Perform all steps on ice or at 4°C Include protease inhibitors in buffers |
| Post-translational modifications are being disrupted during procedure | Include enzyme inhibitors for specific PTMs (e.g. acetylation) if they are relevant to the target protein (e.g. acetylated histone) |
| Samples were over- or under-crosslinked | Optimize incubation time in 1% formaldehyde, usually between 10 and 30 minutes |
| DNA was not sufficiently fragmented, which can result in low precipitation and poor resolution | Optimize sonication parameters and analyze results on an agarose gel. Keep concentration of cells and volume consistent Optimize MNase concentration Samples may have been over-crosslinked; decrease crosslinking time or optimize glycine addition to ensure that fixation is quenched |
| Poor quality DNA gel images | Reduce the amount of DNA loaded onto the gel Run 1-1.5% agarose gel and use a lower voltage (dependent on gel size) |
| Low affinity ChIP antibody | Ensure antibody has been validated for ChIP Try an alternative antibody, if available Try a polyclonal antibody, particularly for X-ChIP in which epitopes may have been blocked Increase antibody incubation time |
| Insufficient ChIP antibody | Use 1-10 µg of ChIP antibody per 25 µg chromatin |
| Insufficient time allowed for antibody-antigen interaction | Perform the immunoprecipitation step overnight at 4°C |
| Specific antibody binding is being disrupted by stringent washes | Do not use a concentration of NaCl higher than 500 mM in wash buffer |
| Beads used were low quality or low affinity | Ensure that Protein A or G is compatible with the ChIP antibody Follow bead product datasheet for optimal volume of beads to antibody ratio Do not freeze beads |
| Incomplete elution | Elute at 65°C Try alternative elution buffers, such as including 1% SDS |
| DNA binding to tubes | Use siliconized or low retention tubes |
