Chromatin Immunoprecipitation Troubleshooting

Chromatin immunoprecipitation protocol

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Immunoprecipitation protocol

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TROUBLESHOOTING

Cross-linking

Too much/little cross-linking

• Under cross-linking can prevent the disassociation of protein-DNA complexes in the following steps and 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.
• If using paraformaldehyde, ensure that it is freshly prepared. 

Chromatin Shearing

Foaming

• Use 1.7 ml microcentrifuge tubes with no more than 400 μl of sample.
• Keep sonicator tip very close to the bottom of the tube.

Under-sheared chromatin

• Perform more shearing replications, turn up the sonication power, cross-link less, or use less cells

Over-sheared chromatin

• Perform fewer shearing replications, turn down the sonication power, cross-link more, or more less cells.

Chromatin degradation

• Samples must be placed on ice in between sonication steps. If the sonication is too long or powerful, unwanted denaturing will take place.

Chromatin Immunoprecipitation

Magnetic beads

• Always fully resuspend beads by vortex before pipetting. Store the beads at 4° C and never allow beads to dry out.
• Check that the subclass of your antibody is compatible with Protein A/G.

Antibody

• Verify that your antibody of interest is ChIP validated.
• Specificity of antibody can be verified by Western blot after IP.
• Too little antibody can result in too little material for successful PCR. Too much can increase PCR background.
• Some antibodies may allow short room temperature incubations with lysate but in general an overnight incubation at 4° C will increase signal and specificity. 

Reverse Cross-linking

Inefficiency

• For most complexes, a 15-minute incubation at 95° C will be sufficient. However, with some samples Proteinase K treatment for 2 or more hours at 62° C may be necessary.
• Initial cross-linking time may also need to be reduced.

DNA purification

Poor yield

• 
  Increase initial cell quantity. If using a commercial purification column, verify that 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.

PCR

High background (high amplification of no antibody control)

• Keep IP buffers cold and increase wash stringency. DNA improperly sheared. Too much antibody or template DNA.

No amplification of product

• Not enough antibody. Verify that your primers are properly designed and that your thermal cycler protocol is agreeable with your Taq master mix. Use more template DNA.