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Does 'verifying by Westernblot after ChIP' really work?'


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#1 muhehehehe

muhehehehe

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Posted 26 May 2014 - 04:41 PM

Does 'verifying by Westernblot after ChIP' really work?'
Hi all:

 I am working on a in vivo Chip-Seq experiment. The protein I am interested in is a transcription factor. I made an N-terminal tagged Knock-in version of this gene(mice), so every protein comes out of this allele is HA tagged. The expression and function of this ha fusion form of protein has been extensively characterize by IHC and Westernblot.

This protein does not express in high level.

Recently I start off working on whether several of my HA antibodies I used can function in ChIP assay. The target of this protein is not known, so I tried to westernblot my Chip sample after final wash to at least confirm my protein is pulled down. However, although all the antibodies I tried worked on regular IP, I can not find my protein of interst in the ChIP-crosslinked pulldown lane.

Before I am going deeper down the troubleshooting route, can anyone give me a suggestion whether the method I used to test the Chipability of my HA antibodies are valid? Although many online protocol simply stated that'To test if the antibody pulls down the target of interest you can perform ChIP up to the final wash after the IP and then boil the beads in loading buffer for 10 min. You can then confirm the presence of your protein in the IP by Western blot.' I rarely find any real paper showing that.

And in my case, is this even possible for low expression proteins? my band is not strong even in regular IP. so is it possible that I actually captured Crosslinked protein but the full length protein is just not enough to show up on westernblot? (I figure the band should be much more faint than the band showed on regular IP)

Thanks alot!!!!
 






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