Chromatin Immunoprecipitation Troubleshooting

Phase

Issue

Recommendations

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 sonications. If the sonication is too long or powerful unwanted denaturing will take place.

Chromatin IP

Magnetic beads

Always fully resuspend beads by vortexing before pipetting. Always store 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.

More Resources:

Chromatin Immunoprecipitation (ChIP) Protocol