ChIP Preparation

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

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.