ChIP for Dummies - (Oct/20/2008 )
Hi all!
For the first time I'm gonna playing a ChiP experiment. I'm looking for MYCN controlled region in some neuroblastoma cell line. I've read in literature that during transcriptional repression MYCN doesn't bind DNA directly but it associate with MIZ-1, which instead binds directly to DNA. Could someone tell me if i can evidence MIZ-1 bounded regions using an antibody against MYCN anyway?
Thanks!
You could not do that that way. You would have to do a first IP with your MYCN antibody, and then anotherone with the MIZ-1. Amplification would then mean association of your 2 proteins at this particular site.
But in generally is it possible to evidence, with a single ChIP, a region not directly bound by MYCN, if it is associated in complex with any other protein bound to DNA?
Not sure I'm getting your question, so I will answer two things ^^
1- I think you cannot rule out the fact, with only one chip, that MYCN does not bind DNA directly but rather binds a protein that binds DNA. In fact, a ChIP alone do not allow you to verify whether your protein is binding DNA by itself, or if it is bound to another protein that binds DNA. The only way to do that is by first crosslinking your proteins and DNA with formaldehyde, do the IP, and then separate the different complexes on a sucrose gradient and then verify if the region you want to check is associated with your protein alone, or with several proteins.
2- If you do the ChIP with your ab, and you see no amplification, then you can conclude that MYCN does not bind this region under those circumstances. More conclusions would need more experiences!
1- I think you cannot rule out the fact, with only one chip, that MYCN does not bind DNA directly but rather binds a protein that binds DNA. In fact, a ChIP alone do not allow you to verify whether your protein is binding DNA by itself, or if it is bound to another protein that binds DNA. The only way to do that is by first crosslinking your proteins and DNA with formaldehyde, do the IP, and then separate the different complexes on a sucrose gradient and then verify if the region you want to check is associated with your protein alone, or with several proteins.
2- If you do the ChIP with your ab, and you see no amplification, then you can conclude that MYCN does not bind this region under those circumstances. More conclusions would need more experiences!
OK!!! thank you for your exhaustive answers!
Ale
I have 3 other questions:
1) What does it mean negative control in a ChIP experiment? I found it's a non immune serum or normal mouse Ig but I don't understand how it works...
2) We used a ARTEK sonic dismenbrator model 150. Is it anyone who could suggest me a protocol of sonication?
3) I found different IP antibodies against human MYCN produced by Santa Cruz. Did you ever use one of these antibodies? If yes, which?
Thanks for your time.
1) Negative control with non immune serum is to ensure that the amplification you get from your ChIP is really specific to the antibody you are using. By using non immune serum, thus a solution that should not immunoprecipitate DNA, you have an idea of the contamination level of your experiments.
2) Never used that, sorry
3) Never used those, sorry 
but you could search the litterature about MYCN to see if anyone has ever done ChIP with this protein, and if so, with whitch company's ab.
2) Never used that, sorry
3) Never used those, sorry
but you could search the litterature about MYCN to see if anyone has ever done ChIP with this protein, and if so, with whitch company's ab.Thnak you very much Madrius..I've also faound normal IgG as negative control. Is It the same thing of non immune serum? Sprry for my stupid question but i never do a ChiP experiment!!!
I think it is, yes.
And never fear of asking questions. We all have to learn 
And never fear of asking questions. We all have to learn
Thank you again Madrius!
