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ChIP Preparation

6 things you should consider before starting your ChIP experiment including formaldehyde cross-linking, chromatin shearing and sonication.

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Considerations before starting ChIP
Always Keep Cells/Tissues on Ice

  • Temperature is critical. Perform cell lysis at 4°C. Keep the samples ice-cold and use ice-cold buffers.

Formaldehyde Cross-Linking

Please Note: Both cross-linking time and formaldehyde concentration are important.

  • When using paraformaldehyde, ensure that it is freshly prepared (final concentration of 1%–1.5%). 

  • Usually, a 10-minute incubation at room temperature in agitation is sufficient. 

  • Under cross-linking can prevent the dissociation of protein-DNA complexes and can result in poor yield. 

  • Over cross-linking can mask epitope sites crucial for antibody binding, prevent proper chromatin shearing, and inhibit the successful uncross-linking of the complex in subsequent steps.

 
Chromatin Shearing and Sonication

  • Make sure the sonicator probe is not in contact with the tube wall. 

  • Increase the number of sonication steps; however try to avoid increasing the time (or the power) of each step as this may overheat the sample and lead to the 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). 

  • Different cell types may have different optimal DNA fragmentation. Determine appropriate sonication times to get your optimal DNA fragmentation.

 

IP Fraction, The Right Choice Of Beads & Primary Antibody

Beads

  • 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.

Antibody

  • Insufficient antibody can result in too little material for successful PCR analysis.

  • Too much antibody can increase PCR background.

 

Reverse Cross-Linking

Usually a 15-minute incubation at 95°C is sufficient. However, with some samples, Proteinase K treatment for 4 or more hours at 65°C may be necessary.

 

DNA Elution & Purification

  • 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 wash will inhibit elution. 

  • Make sure the elution buffer is placed directly onto the silica membrane and allowed to adsorb for at least 1 minute.