detecting DNA-protein interaction - (Oct/31/2013 )

HI,

So I have a protein which is translocated into the nucleus when transfected in HeLa cell. But I have no idea as such about the protein. But since it translocates inside the nucleus the question that arises is whether it can bind to DNA, individually or in a complex with other protein. But since I have no idea of any putative DNA sequence that can possibly bind to it, I cant think about CHIP or EMSA or any other molecular biology based technique.

It would be of great help if anybody can suggest me any option ,biophysical or mol. bio based, which can confirm if the protein atall binds to DNA. And then if it binds to DNA I can surely do the CHIP-seq to identify the proper sequence in which it is binding.

Thanks

Sayantani

You could randomly label your DNA and pull down with an antibody. This should tell you whether or not you have any DNA present.

jerryshelly1 on Thu Oct 31 15:33:37 2013 said:

You could randomly label your DNA and pull down with an antibody. This should tell you whether or not you have any DNA present.

Hi Jerry thanks a lot for replying...I am so new to post in this forum and glad to see that you readily helped. Thanks

Can you kindly elaborate a little more on the protocol of the experiment?How will i Label the DNA and what are the techniques to pull it down?

awaiting your reply.

I don't have a protocol that is easily accessible, but I am sure you can google one or someone on here will be happy to contribute one.

This experiment would essentially be an IP. You would obtain a cell extract and add random DNA oligo's that are radiolabeled. Incubate the mixture to allow proper DNA-Oligo binding. Wash your sample and then add a specific antibody that can recognize your protein. Incubate an pull down using magnetic or agarose beads. You could then run a gel and detect any radioactivity on autorad film. If you see a band(s) you would know if your protein or multiple proteins are bound.

Does this make sense?