ChIP Preparation
Considerations before starting ChIP
Always Keep Cells/Tissues on Ice
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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.
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When using paraformaldehyde, ensure that it is freshly prepared (final concentration of 1%–1.5%).
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Usually, a 10-minute incubation at room temperature in agitation is sufficient.
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Under cross-linking can prevent the dissociation of protein-DNA complexes and can result in poor yield.
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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
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Make sure the sonicator probe is not in contact with the tube wall.
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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.
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Add ice to the sonicator to avoid the sample overheating.
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Preferably do not sonicate chromatin to a fragment size of less than 500 bp (perform the size-testing step).
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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
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Always fully resuspend beads by vortexing before pipetting.
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Always store at 4°C and never allow beads to dry out.
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Check the subclass of your antibody is compatible with Protein A/G.
Antibody
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Insufficient antibody can result in too little material for successful PCR analysis.
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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
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Use different washing buffers (low & high salt, LiCl, and TE buffers).
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While using a commercial purification column, check the column is completely dry after the wash step as any leftover wash will inhibit elution.
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Make sure the elution buffer is placed directly onto the silica membrane and allowed to adsorb for at least 1 minute.
