Possible Causes

What You Can Do?

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Not enough cells/chromatin

Add enough chromatin for each IP experiment. We suggest using at least 25 μg of chromatin for each IP.

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Incorrect Protein A/G/L used

Make sure that the Protein A/G/L beads are capable of binding to the antibody subclass being used.

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Cross-linking process too long

Over Cross-linking with formaldehyde might mask the antibody binding sites and reduce antibody binding ability. It is advisable to optimize the cross-linking steps by using different concentration of formaldehyde or reducing cross-linking time.

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Not enough antibody

Titre antibody amount used for each IP to determine the optimal condition. Up to 10ug of antibody can be used for each IP experiment.

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Washes too stringent

Reduce the number of washes. Reduce salt concentration in the wash buffer.

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Antibody not capable of
immunoprecipitation

Try a different antibody. Try polyclonal antibody if monoclonal antibody does not work well.

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The chromatin size might be too small

Make sure that the shearing condition is not too harsh which might results in fragments of DNA smaller than what the primers are able to amplify.

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Incomplete elution from the Protein
A/G/L beads

Incubate beads in elution buffer at 65°C with frequent mixing.

Possible Causes

What You Can Do?

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PCR reagent might be contaminated

Prepare new solutions from stock.

Possible Causes

What You Can Do?

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Inadequate washing

Use a more stringent washing buffer. Try to  use a high salt washing buffer or increase the number of washes.

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Non specific binding to Protein A,G or L

Include a pre-clear step by incubating lysate with Protein A/G/L agarose beads.

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Too much DNA template added
to the PCR reaction, or too many
cycles of amplification

Add less DNA template or reduce the number of cycles of amplification.  Alternatively, real-time PCR can be used for the detection of ChIPed DNA products.

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Buffers may be contaminated

Use freshly prepared lysis or wash buffers.