5 replies to this topic

[molbio]

Hi,

I want to study histone modifications affecting a particular gene that I'm working with. I guess ChIP is the method to use, but I (or my lab) have no experience in this method. How hard is it to do, how much time should I expect for this kind of experiment? Any advices? Are there any companies that do it for you?

Thanks!

[pcrman]

I must say ChIP is quite challenging. It usually takes 2-3 days besides the time needed for cell treatment. I don't know if there is any company offering such service but Upstate does sell good ChIP kit. Also check their website for more info on ChIP such as protocol, faqs.

[methylnick]

Hi Molbio,

may the ChIP gods look upon you favourably.

Pcrman is right in saying it is a very challenging technique to master, but once mastered it's very rewarding to see the results.

Upstate have a very good kit, I would also like to add that Abcam are very good with their antibodies, some if not most histone modification antibodies are guaranteed for ChIP.

I know abcam have sourced their H3K9 trimethylated from the Jenuwien Lab, a very good antibody for heterochromatin.

Be aware with the antibodies though, even though they may well be guarnteed for ChIP I have experienced that this is not the case, so don't go out and buy a big batch of a particular antibody until you are sure the darn thing works! Note the bacth number of that antibody that you test and insist on getting that batch as the activity can vary between batches.

good luck with it and may the ChIP-force be with you always!

Nick

[molbio]

Thanks for our answeres. I'm going to try it at least...
I´ve been looking into some antibodies. I suspect that my gene of interest is deacetylated (due to TSA studies) do I have to try H2A, H2B, H3 and H4, or is one of these sites more likely to be acetylated, or is all of them acetylated if one is...
Could you recommend which one to start with?

Thanks again!

[pcrman]

H3 and H4 are most studied for acetylation. Try them first.

[pcrboy]

The part that is gives people most headache (and sometimes not in your control) is the specificity of the antibodies. Before doing ChIP, I would run a western from your antibody with the appropriate controls.