Chromatrap®’s ChIP kits provide a quicker, easier and more efficient way of performing ChIP assays. However, there are a number of steps you can take to ensure positive ChIP results. Here is our top ten:
1. Chromatin quality is crucial for successful Chromatin Immunoprecipitation.
ChIP assay success is reliant on the quality of chromatin prepared, with lysis, fixation and shearing being the three most important aspects requiring optimisation. Poor chromatin = poor ChIP result.
2. Keep chromatin frozen
When stored or handled at room temperature, chromatin will degrade swiftly. In addition to using small aliquot volumes of chromatin, ensure it is kept on ice throughout the experiment and avoid repetitive freezing and thawing.
3. Optimise your cell fixation
Ensuring your fixation times are optimised is crucial to the preservation of the chromatin structure during isolation and ChIP. Too long and cells can become resistant to lysis and any subsequent shearing, whereas fixation times that are too short will result in poor cross-linking efficiency and DNA loss following immunoprecipitation.
4. Use fresh fixation solution
Using fresh, methanol-free formaldehyde in chromatin preparation will result in more reproducible results and increased fixation.
5. Choose an appropriate shearing method for your cells
Provided the temperature is controlled during the sonication process and emulsification is avoided, good quality chromatin can be obtained from most cell types using this method.
Enzymatic shearing is useful if a sonicator is not available and is less disruptive to the epitopes of the protein of interest recognized by the specific antibody. Enzymatic shearing is essential when carrying out native ChIP as sonication can disrupt the protein/DNA complexes.
Certain cell types may be resistant to lysis resulting in poor enzymatic shearing efficiency, in this instance try sonication.
6. Ensure chromatin is sheared to between 100-500bp
In order to avoid reduced ChIP efficiency, it is crucial to check the chromatin is sheared to fragments between 100-500 bp when preparing chromatin.
Over-shearing of chromatin will result in very small fragments which can reduce primer recognition and lower PCR efficiency. Over shearing by sonication can also increase the risk of damaging the protein epitopes.
Under-shearing will produce larger fragments which will increase non-specific binding in the ChIP assay.
7. Always use a ChIP validated antibody
Unvalidated antibodies do not always work well in ChIP, so the use of high quality and specific ChIP validated antibodies is essential, as antibodies must recognise and bind to native protein that is bound to DNA.
8. Always run a positive and negative antibody control
Both positive and negative controls should be run alongside any test antibodies, as this will indicate the efficiency of the immunoprecipitation. Additionally, positive and negative gene targets are also good controls to ensure antibody enrichment is selective.
9. Get the ratio of antibody to chromatin right to avoid compromising the signal to noise ratio
Unspecific binding can result from antibody oversaturated ChIP assays, whereas too little antibody will not bind the chromatin present resulting in a poor sample representation.
Due to Chromatrap’s® unique solid phase platform a greater surface area for antibody binding capacity is provided, which promotes molecular mixing and minimises non-specific background and is typically more efficient than magnetic and agarose beads.
10. Optimise lysis buffers for lysing of cells in ChIP assay
Always pre-warm lysis buffers to 40°C with occasional mixing to remove any precipitates, such as mild detergents. Ensure the buffer is returned to room temperature for the lysis step and all precipitates are re-dissolved. Ensure lysis buffer volumes are optimised for each chromatin preparation before proceeding with your ChIP assay. Lower cell numbers (1-5 million) require less lysis buffer than greater cell numbers (10-15 million).