Does my protein bind nucleic acids? - (Nov/26/2008 )

I am working on a nuclear protein.
Are there tests I can do to see whether my protein can bind or interact with DNA (or even RNA)?
Thanks.

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My old PhD lab developed a series of probes that represented every 5nt combination. You use them in a gel-shift (aka EMSA) assay. Here's my PI's page.

http://www.mmb.usyd.edu.au/research/academ...ofiles/joel.php

You could also try a DNase footprinting assay. Some might say it's a bit old-school, but it will do the job.

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Hallo if you send mi your mail I will send you link where you will by able to download pdf book about method for DNA Protein interaction

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But how can I perform the footprinting if I don't know the target DNA?

One way I was thinking is the following.

1. Extract my protein.
2. Treat a little bit of DNAase.
3. Purify DNA.
4. Poly-A extension.
5. Add oligo dT and extend.
6. Do PCR with oligo dT.
7. Clone it by blunt-end ligation.
8. Seqence the clones.

What do you guys think?

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Take your gDNA and cut it with a 4-base cutter. The DNA fragments need to be 100-300 bp in length or the DNA is too large and the complex starts to lose stability

If you have access to chromatography equipment,
Mix the cleaved DNA with your protein and incubate. As a starting point, I'd suggest a phosphate buffer, say 50 mM, fairly neutral pH, with 150 mM NaCl.
Run some of your protein only through a gel filtration column and note the elution profile.
then run some of the complex. collect the fractions containing your protein. If there's any DNA interactions, it will accelerate the protein's migration.
Clone this. Do the DNase footprinting experiments.
Write up a wonderful paper, and tell everyone how great the forum is.

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