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Check the possible causes and find out the solution!
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
immunoprecipitationTry 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 beadsIncubate beads in elution buffer at 65°C with frequent mixing.
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Check the possible causes and find out the solution!
Possible Causes
What You Can Do?
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PCR reagent might be contaminated
Prepare new solutions from stock.
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Check the possible causes and find out the solution!
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 amplificationAdd 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.