how everyone obtain skills for chip assay? - Is it necessary to learn it from another lab? (Jul/05/2006 )

Hello, everyone, I've been reading posts in this forum about CHIP assay and benefit from you, especially about the volume of sonication(should be less than 400ul). However, in our lab the CHIP assay still doesn't work very well, at least it failed to confirm my PI'S hypothesis. I think probably the primers do not work well, though we already have designed and tried 4 pairs of primers in promoter region. In one word, we think learning how to do chip assay in another lab might work. There is another lab, which is 4 hours ride from our university, they work with chip in the same field as us, and they have been publishing papers in an amazing speed recently. My PI call the professor and get a very positive response. However, after that, both my PI and I failed to get any feedback from the professor about arranging the details and schedules of visiting their lab......
We are desperate to make it work, because it is really important for the project, now, I hope to get advices. Maybe I just has not tried enough, or I can try the fast chip protocol recommended in this forum......
BTW, before me, a senior graduate student worked on CHIP in our lab for over a year. Besides, we are mainly working on very confluent cells(15day culture), maybe that is why it's hard to make it work, but people have published paper using overconfluent cells.
I appreciate any input, thanks a lot!

We need more info! Which protocol are you using? Can we see it? That's just for a start!
Do you know anything about the protein you are IPing? Is it regulated according to the confluency of the cells? Is your Ab working? Can you amplify your target gene in the input chromatin? Are you sure you are sonicating the DNA in the right way? 400ul doesn't mean anything: the sonication efficiency depends on the sonicator, on the power you are using...
Can you please give us more info?

Thank you for your quick reply. The main protocol I've been using is from upstate and other labs. We start from acetyl-H3 antibody from upstate, which is established to be related to activation of gene expression. A few years ago, people had published that alpha1 anti-trypsin was a gene that was highly expressed in postconfluent cells and using chip assay, they show that the promoter region was associated with acetyl-H3 in postconfluent cells. I'm also working on different cell stages(proliferating and postconfluent cells),and we know another gene that is very highly expressed in postconfluent cells and we are trying to find the promoter sequence that is associated with the acetyl-H3 and we expect a difference between postconfluent cells and proliferating cells. The acetyl-H3 antibody works because I amplify anti-trypsin promoter region and I got a enrichment of the promoter region with antibody each time, though not that huge difference as people published. I also amplified the unrelated region of anti-trypsin gene and there is no enrichment with acetyl-H3 antibody compared with rabbit IgG. \ I have had no problem in amplifying target gene in input chromatin.\ After sonication I ran gel and I think the quality is fine.
Attached are the sonication DNA gel pictures and protocol I'm using.
Thank you very much!

We need more info! Which protocol are you using? Can we see it? That's just for a start!
Do you know anything about the protein you are IPing? Is it regulated according to the confluency of the cells? Is your Ab working? Can you amplify your target gene in the input chromatin? Are you sure you are sonicating the DNA in the right way? 400ul doesn't mean anything: the sonication efficiency depends on the sonicator, on the power you are using...
Can you please give us more info?

picture of sonicated DNA

The protocol you are using looks very similar to the one I use. If I understood correctly, you are saying that you can amplify the input chromatin using the primers you designed, that should amplify whatever you IP. If this is true, I would definitely exclude that your primers are not working.
Just a stupid question: did you design your primers in a way they'll amplify a region of 200-400bps? Your chromatin is sonicated so that you have less than 300 bps fragments. I do it in a way I have 500bps. Are you sure you are using enough Ab? like 3ug for 10^7 cells?

Your suggestion is very helpful.
I'm happy to know that the protocol is similar. For your question, we did use primer amplifying a region of 200-400bps. Probably I should try to sonicate the chromatin to the size you did, or design primers amplifying shorter regions.
For the antibody, I measured DNA concentration after crosslinking, and use 20ug chromatin with 3ug acetyl-H3 antibody from upstate. For the postconfluent cells(15 day culture), it's impossible to count cell numbers.
Thank you very much for your help.