Does 'verifying by Westernblot after Crosslinking really work?'

[muhe1985]

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 mice, so every protein comes out of this allele is HA tagged. The expression and function of this tag 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!!!!

[muhe1985]

Really appreciate some comments on this topic!!! Thanks!!

[kerplunk412]

I spoke with someone who got her PhD in a lab that did a lot of ChIP, and their protocol was to heat the sample at 95 degrees for 30 minutes in loading buffer before loading for Western blot. You may want to try that.

You may also want to try saving your washes and running some on Western blot to see if you are losing your protein if interest in a wash step.

In general, I believe your methods are valid. I have done many RNA-binding protein IPs, and I was always able to confirm the success of the IP by Western blot, often with as little as 1/20 of my sample, although the proteins were reasonably well expressed.

I personally would not trust ChIP-Seq data without verification by Western blot, although I guess many do.

[muhe1985]

Thank you very much for your detailed reply and instruction!

According to your experience, what would the band from CHIP-Westernblot look like? a faint 'smear like' band or a solid strong band?

Best,

I spoke with someone who got her PhD in a lab that did a lot of ChIP, and their protocol was to heat the sample at 95 degrees for 30 minutes in loading buffer before loading for Western blot. You may want to try that.

You may also want to try saving your washes and running some on Western blot to see if you are losing your protein if interest in a wash step.

In general, I believe your methods are valid. I have done many RNA-binding protein IPs, and I was always able to confirm the success of the IP by Western blot, often with as little as 1/20 of my sample, although the proteins were reasonably well expressed.

I personally would not trust ChIP-Seq data without verification by Western blot, although I guess many do.