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Low signal in ChIP

Explore causes of low signal in ChIP

Low signal

Possible causes

Solution

The chromatin size may be too small

Sonication to a fragment size of less than 500 bp can displace nucleosomes, as intranucleosomal DNA becomes digested.

  • Do not sonicate chromatin to a fragment size of less than 500 bp. 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

Excessive cross-linking can reduce the availability of epitopes and thus reduce antibody binding.

  • 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.

Not enough starting material

  • We suggest using 25 μg of chromatin per immunoprecipitation.

Not enough antibody included in the immunoprecipitation

  • We suggest using between 3-5 μg of antibody in the first instance. This could be increased to 10 μg if no signal is observed.

Specific antibody binding is being eliminated

High NaCl concentration in the wash buffers can be too stringent and remove specific antibody binding.

  • Do not use higher than 500 mM NaCl in your wash buffers.

Cells are not effectively lysed

  • We suggest using RIPA buffer to lyse cells.

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

Cross-linking may be required to stop proteins dissociating from the DNA. Therefore X-ChIP is more suitable when analyzing proteins that have either a weaker DNA affinity or are a long way from DNA. Histones are tightly associated so N-ChIP can be performed when studying histones.

  • Use X-ChIP for proteins with weaker DNA affinity or that area long way from DNA.

Monoclonal antibodies may not be suitable for X-ChIP

The epitope may have become masked during cross-linking thus preventing epitope recognition.

  • We suggest using polyclonal antibodies that will recognize multiple epitopes as there is an increased chance of immunoprecipitating the protein of interest.

The wrong antibody affinity beads were used

Protein 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 suggest using a mix of Protein A and Protein G that have been coupled to sepharose.