anyone else use chromatin immunoprecipitation in their research? just curious if anyone has any unique tips or suggestions.

I'm doing ChIP assaysquite frequently, with both cell lines and animal tissues. The antibodies against modified histones which I've been using work beautifully, but I've been having some trouble when using antibodies against transcription factors. In my hands, the critical step is sonication, and also the selection of adequate positive and negative controls to test the results.

i am using a transcription factor and use acetylated histone as a control. then use known targets and know not-target genes to test the specificity of the IP. i'm only using tissues, no cell culture. i definitely found that optimizing the sonication was important.

Just out of curiosity, nin1318, you said you're working with tissues: do you work with the whole tissue, or do you isolate the cells? In case you're working with whole tissues, how do you do the fixation, by perfusion, by mincing the tissue with a razor blade, by other method...?
Thanx. Up to now I've worked with tissues which I can perfuse and disssagreggate with collagenase prior to fixation. Now I'm interested in working with small amounts of tissues (from embryos) which I can not perfuse, and I'm trying to figure out which may be the best protocol.
Cheers!

well i was following a modification of a protocol i read from a paper..i don't remember it off the top of my head but i'll look around for it. i use whole tissue; i used to slice the (mouse brain) tissue with a McIlwain (sp?) tissue chopper into 500um slices, then put them in the fix. however i found that i can make slices just as well if not better using a razor blade or scalpel. basically i follow the upstate protocol from their kit, except for the slices of tissue and then stopping the fix reaction with 1.2M glycine.

as long as you don't put too much in the tube at once before sonication, you should be alright as far as sample loss goes. even if it foams up, the sample should still be sheared. you need to optimize it by having multiple sample aloquots and sonicating them for different lengths of time or at different powers, and then running it out on a gel to see how the shearing is working (if your fragments are in the range you would like)

We specialized in ultrasonic technology - especially for homogenizing and cell disruption - since more than 45 years.

Since longer time, we sold a lot of euipments worldwide for ChIP and we never had any negative feed back because of high standard of our units. The reproduceability is perfect and the power density (not the electriy power of the power supply!!) is high enough for this task.

The main issue is the perfect unit, there is a wide gap between ultrasonic devices, not only regarding the manufacturer, but also, and especially regarding the correct tool to use - combined with applicational hints from specialists!

For any details, do not hesitate to contact me