You can use toluidine to stain DNA in PAA gel. The protocol is the same like for radiolabeled DNA but you have to use grater amount of DNA (pmoles).

https://www.researchgate.net/post/Electrophoretic_mobility_shift_assay_EMSA_used_to_measure_DNA_binding_activity_requires_radioactivity_Is_there_a_non-radioactive_approach

I used Cy5 labeled DNA for non-radioactive EMSA. The results were very comparable to P32 labeled DNA. You need a bit more DNA for detection, depending on the sensitivity of your imaging system, but you do not need to care about half-lives of P32 anymore!

I'm a bit biased, I know, but I would prefer to use MicroScale Thermophoresis (MST) instead. With this technique, you will not only see IF molecules interact, but you can also determine binding affinities!

Here is a 3min video to explain the technique:

http://www.nanotemper-technologies.com/news-events/news/article/watch-a-3-minutes-movie-to-explore-microscale-thermophoresis-mst/

Please feel free to contact me for any questions on the technique and how it might help you with your protein-DNA interaction study!

Hi Heide,

I am planning to check one of my protein for DNA binding function. I have seen the protein localisation into the nucleus. I want to do some sort of basic experiment to get a hint if protein is actually binding to DNA.

Do you have any suggestions?

I am thinking of incubating purified protein with gDNA (not sure about conditions).

If you need a simple yes/no answer you can even stain gel with ethidium bromide and visualize it under UV.

Yes, thats what I am going to try to get a hint.

But, my protein is in PBS, Do I need to exchange buffer of my protein?

Thanks for your suggestions.

You can set up few emsas with protein in PBS to get fast answer. But if you will be unable to see shifted bands exchange buffer to proper one and repeat experiment to exclude possiblle buffer interference into DNA/protein interaction.

Ok. Thank you..

I guess PBS is fine to start with; I agree with Leszek that for Protein-DNA interaction you might need to play around with pH and salt conditions. Usually, pH7 and 100mM NaCl is suitable, for EMSAs and MST.

I am also planning to conduct non-radioactive EMSA to determine if the regulatory proteins that I am studying can bind to my test promoters. I will just synthesize the promoters and purify my proteins and run it in a gel.  In this case, I will use ETBR and dye to see the bands. Is this advisable? If you can suggest, it will be a great help for my experiment. Thank you in advance!