Chromatin Immunoprecipitation (ChIP) overview
In view of the increasing evidence that chromatin structure plays a decisive role in regulating gene expression, there is an increasing tendency to simultaneously study both structural and functional aspects of gene regulation. Below is a summary of the most relevant tips to help you optimize your ChIP experiment.
Temperature is critical. Perform cell lysis at 4 °C. Keep the samples ice-cold and use ice-cold buffers.
Both cross-linking time and formaldehyde concentration are important.
Please note: When using paraformaldehyde, ensure that it is freshly prepared (final concentration of 1%–1.5%).
Under cross-linking can prevent the disassociation of protein–DNA complexes and result in poor yield.
Over cross-linking can mask epitope sites crucial for antibody binding, prevent chromatin shearing, and inhibit the successful uncross-linking of the protein–DNA complex.
Avoid large fragments in the tissue suspension.
Pipette with cut tips for better homogenization.
Ensure the sonicator probe is not in contact with the tube wall.
Increase the number of sonication steps; however, avoid increasing the time (or the power) of each step as this may overheat the sample and lead to loss of antigenicity.
Add ice to the sonicator to avoid the sample overheating.
Preferably do not sonicate chromatin to a fragment size of less than 500 bp (perform the size testing step).
Please note: Different cell types may have different optimal DNA fragmentation. Determine appropriate sonication times to obtain optimal DNA fragmentation.
Do not forget about the input sample.
IP fraction represents the fraction of diluted and pre-cleared ChIP. It is used for the immunoprecipitation step involving the antibody of interest or control antibody.
Always fully resuspend beads by vortexing before pipetting.
Always store at 4 °C and never allow beads to dry out.
Check the subclass of your antibody is compatible with Protein A/G.
Verify your antibody of interest is ChIP validated. (Specificity of antibody can be verified by Western Blot after IP.)
Insufficient amount of antibody can result in not enough material for successful PCR analysis.
Too much antibody can increase PCR background.
Some antibodies may allow short room temperature incubations with lysate; however, in general, an overnight incubation at 4 °C will increase signal and specificity.
Use non-immune IgG in the IP incubation mix from the same species the antibodies were produced in.
Incubate IP fraction with beads (without antibody coating).
Usually, a 15-minute incubation at 95 °C is sufficient. Some samples require Proteinase K treatment for four or more hours at 65 °C.
Use different washing buffers (low & high salt, LiCl and TE buffers).
While using a commercial purification column, check the column is completely dry after the wash step as any leftover moisture will inhibit elution.
Make sure the elution buffer is placed directly onto the silica membrane and allowed to adsorb for at least one minute.
Prepare new solutions from stocks to avoid contamination.
A weak PCR signal or no DNA amplification shown in the samples may be due to an inadequate primer result in the PCR amplified region spanning the nucleosome-free region.
Please note: Test primer pairs for genomic DNA using different primer conditions and dilutions. The use of standard/input DNA will determine the efficacy of the primers.
Ensure there is enough DNA in the PCR reaction and your PCR conditions are optimal. Add more DNA to the PCR reaction or increase the number of amplification cycles if needed.
To avoid variations between replicates, add the same amount of protein G/A-agarose or magnetic beads for all samples. Ensure beads are well suspended while pipetting.
While there is no product in the positive control ensure there is enough chromatin or antibody within the IP reaction. Ensure the IP incubation time is optimized.
Complete the elution of chromatin from protein G/A beads. Elution is optimal at 65 °C with frequent mixing to keep beads suspended in solution (~10 minutes).