ChIP'ing for Big, Indirect Coactivators - Looking for tips on how to get this difficult ChIP to work.. (Sep/29/2007 )
I am looking for some tips / feedback on how you successfully ChIP for huge proteins that are not direct DNA binders (maybe this is really two questions: 1) howto big proteins? 2) howto proteins that are indirectly associated with DNA).
I'm working with a 220kd protein that binds many secondary factors that are in turn associated with additional factors that directly bind DNA. ChIP'ing for this protein is turning out to be exceedingly difficult. In general, I have relatively little problems ChIP'ing for other proteins, so lets just assume that i "know" how to do ChIP in general. I've tried 3 different antibodies for the same protein thus far, but it's been 4 months now and my ChIPs continue to fail to produce enrichment for this protein in the genomic location of interest. However, a previous post-doc in our group was able to do this ChIP "without much trouble".
any tips you might have would be very much appreciated.
I'm working with a 220kd protein that binds many secondary factors that are in turn associated with additional factors that directly bind DNA. ChIP'ing for this protein is turning out to be exceedingly difficult. In general, I have relatively little problems ChIP'ing for other proteins, so lets just assume that i "know" how to do ChIP in general. I've tried 3 different antibodies for the same protein thus far, but it's been 4 months now and my ChIPs continue to fail to produce enrichment for this protein in the genomic location of interest. However, a previous post-doc in our group was able to do this ChIP "without much trouble".
any tips you might have would be very much appreciated.
I don't know if you've seen this reference or not but I think it would apply.
BioTechniques. Nov 2005. Volume 39(5): p 715-725
They use a two-step crosslinking method for ChIP. The first step stablizes protein-protein interactions and the second step is the typical formaldehyde crosslinking. I've never tried it but it at least looks relevant.
Sweet! I'll try it. Thanks for the reference. It will be good foder for my blog too! This experiment is killing me! 
Hi,
this will be then an stupid comment but have tried longer crosslinking and adding more antibody 
this will be then an stupid comment but have tried longer crosslinking and adding more antibody
Hi Zek.. not a stupid comment, it's good to cover ground. I have tried both of those options. Longer crosslinking is always a trade off though between capturing 2º and 3º interactions and sensitivity of your antibody. The longer you formalin treat, the more you will distort your epitope of interest and likely reduce your antibody specificity.
More antibody. I've tried that, but it also did not work that well, but i only increased it by 3x. Maybe I should try 10x more antibody.
Hi,
thank you Jonathanjacobs. I will keep this in mind 
Would you update your results if you get something new?
Cheers
this will be then an stupid comment but have tried longer crosslinking and adding more antibody
Hi Zek.. not a stupid comment, it's good to cover ground. I have tried both of those options. Longer crosslinking is always a trade off though between capturing 2º and 3º interactions and sensitivity of your antibody. The longer you formalin treat, the more you will distort your epitope of interest and likely reduce your antibody specificity.
More antibody. I've tried that, but it also did not work that well, but i only increased it by 3x. Maybe I should try 10x more antibody.
can you run some of your crosslinked lysate on a western maybe even just a dotblot to get an idea if your anitbody will still recognise the epitope?