"Help" : how to identify of proteins binds to specific DNA - (May/29/2005 )
Hello, all of you.
I have had a headache.
I got a problem in my research.
I wanna identify proteins that binds to specific DNA sequence. For example, promoter region.
After searching that issue, I could not find a solution.
One solution on this problem is using synthesized DNA column.
After making the column of my DNA sequence, load the mammalian cell lysate.
However this system is artificial method. So I wanna in vivo method.
Anyone have some idea, reply or contact to me.
Thank a lot.
Have a nice day.
I have had a headache.
I got a problem in my research.
I wanna identify proteins that binds to specific DNA sequence. For example, promoter region.
After searching that issue, I could not find a solution.
One solution on this problem is using synthesized DNA column.
After making the column of my DNA sequence, load the mammalian cell lysate.
However this system is artificial method. So I wanna in vivo method.
Anyone have some idea, reply or contact to me.
Thank a lot.
Have a nice day.
Hi,
If am not wrong in understading your msg, you want to identify all proteins that bind to a specific DNA sequence?
In this case, I think, it's hard to think of an in-vivo approach. Instead you should try "fishing the ligands" (in your case, it's proteins) using BIACORE. First immobilize the DNA of your interest onto the flowchip then inject the cell extract onto it. Definitely you will see the binding if any. To identify the binding proteins, you can employ an additional Maldi-tof MS. You can find detail information on BIACORE application notes.
If you have got a better idea, please share with us!
Goodluck!
Le
hi
i've not done this but i think the best one would be chromatin immunoprecipitation? 
Use Yeast One-hybrid system to screen binding candiates from an expression library. This is the only in vivo method but it may not reflect the real DNA-protein interaction in mamanlian cells. BTW, many false clones will be screened so you have to confirm the binding using in vitro assay (EMSA, UV-crosslinking etc.).