I am looking for advice on which epitope tags and antibody combinations people have successfully used to generate ChIP-seq quality pull-downs. I have already tried the HaloTag system from Promega which does not work for my TF of choice so am looking to add a smaller tag to my TF under the control of an inducible promoter to achieve native expression levels. From looking on-line I have seen mixed reviews for several tags (Specifically HA, His, Ty1 and V5) but no specific information on the antibodies/capture being used. Additionally, I thought that a TAP capture method may offer better signal to background ratio than standard immunoprecipitation but there is little evidence of this being widely used which has me questioning it's effectiveness for ChIP-seq application.
It would be very valuable to me if people could share their real lab experience of using tagged protein ChIP-seq and what has worked for them.
Dear Louise,
I would recommend using HA tag for Transcription Factors and untagged version of strain or cells lines can be used as control. But make sure the tagged version of TF is functional under the conditions in which you are growing the cells lines.
For V5 tag I observe nonspecific binding and that might cause lot of problem .
Thanks Prashant,
That is very useful to know that V5 has high background in ChIP-seq applications.
With regard to the use of the HA tag, which antibodies have you had success with? I can see there are several advertisted as ChIP quality but these may not all be of ChIP-seq quality so it would be good to know specifically which of these you have has success with.
Dear Louise,
I am after the same question. Were you able to find a nice ChIP-Seq ab for your HA tag? Otherwise I read Sigma is doing V5-beads so V5 might be a good alternative. What's your thoughts on that? Thank you!
Hi,
I just tested anti HA from MBL (561) and it seems quite unspecific. Trying once more and then moving on to Cell Signaling #3724.
Active Motif recently introduced their Tag-ChIP-IT Kit which utilizes a tag specifically designed to work in ChIP. The tag is much smaller than the HaloTag system, and provides more reliable results than FLAG or HA tags. You can clone the tag sequence into any expression vector and use the Tag-ChIP Kit, which includes an antibody directed against the tag, for the pull down. The enriched DNA is high quality and can be used for ChIP-seq.
The AM tag for Tag-ChIP-IT Kit is used for mammalian system, can it be used in bacteria system?
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