ChIP
troubleshooting tips

Non-specific binding to Protein A or G beads
Include a pre-clearing step, whereby the lysed sample is mixed with beads alone for 1
hr and removed prior to adding the antibody.
The ChIP buffers may be contaminatedPrepare fresh lysis and wash solutions.
Some Protein A or G beads give high backgroundSome Protein A or G beads can give high background levels. Find a suitable supplierthat provides the cleanest results with low background in non-specific control.

Low resolution with high background across large regions
DNA fragment size may be too large
DNA fragmentation should be optimized when using different cell types. Bothsonication times and enzyme incubation times can vary. We would suggest a DNA
fragment size of no larger than 1.5 kbp. If chromatin is being digested using enzymes,mononucleosomes (175 bp) can be obtained.

Low signal
The chromatin size may be too small
Do not sonicate chromatin to a fragment size of less than 400 bp. Sonication to asmaller size can displace nucleosomes as intranucleosomal DNA becomes digested. If
performing N-ChIP enzymatic digestion is generally sufficient to fragment chromatin.
If performing X-ChIP, the cells may have been cross-linked for too long
Cross-link with formaldehyde for 10-15 min and wash well with PBS. Cells may need to
be treated with glycine to quench the formaldehyde. Excessive cross-linking canreduce the availability of epitopes and thus reduce antibody binding.
Not enough starting material
We would suggest using 25 μg of chromatin per immunoprecipitation.

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Not enough antibody included in the immunoprecipitation
We would suggest using between 3-5 μg of antibody in the first instance. This could beincreased to 10 μg if no signal is observed.
Specific antibody binding is being eliminated
Do not use higher than 500 mM NaCl in the wash buffers as this may be too stringent
and remove specific antibody binding.
Cells are not effectively lysed
We would suggest using RIPA buffer to lyse cells. The composition can be found in our
X-ChIP protocol.
No antibody enrichment at region of interest
The epitope is not found at the region of interest. Be sure to include a positive control
antibody to confirm the procedure is working well e.g. H3K4me3/H3K9me3 antibody
at active/inactive promoters.
N-ChIP is not suitable
X-ChIP may be more suitable when analyzing proteins that have either a weaker DNA
affinity or are a long way from DNA. Cross-linking may be required to stop proteinsdissociating from the DNA. Histones are tightly associated therefore N-ChIP can be
performed when studying histones.
Monoclonal antibodies may not be suitable for X-ChIP
The epitope may have become masked during cross-linking thus preventing epitope
recognition. We would suggest using polyclonal antibodies that will recognize multiple
epitopes as there is an increased chance of immunoprecipitating the proteins oninterest.
The wrong antibody affinity beads were usedProtein A and G are bacterial proteins that bind various classes of immunoglobulins
with varying affinities. Use an affinity matrix that will bind your antibody of interest. We
would suggest using a mix of Protein A and Protein G that have been coupled tosepharose.

Problems with PCR amplification of immunoprecipitated DNA
High signal in all samples after PCR, including no template control
Real-time PCR solutions may be contaminated, we suggest preparing new solutions
from stocks.
No DNA amplification in samples
Be sure to include standard/input DNA to confirm that the primers are working well.
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