A different approach to use is fluorescence polarization/anisotropy. Purchase short oligonucleotides with a fluorescence tag (fluorescein) then monitor binding of your proteins. This approach is very sensitive. Some plate readers can perform polarization measurements allowing high-throughput screening.

I have used biotinylated DNA sequence and streptavidin beads to study protein-DNA interaction . Although there is some loss of bound protein during washing steps, it is pretty quantitative and can give a good idea about what DNA sequence/ or which protein can interact with each other.

SPR can be used to get a quantitive binding assay and to footprint the exact region of unlabeled protein binding. See the links below for the methodology and a company that runs it as a service.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3737563/

http://www.inspiralis.com/go/redcat_chip.php

You could do MicroScale Thermophoresis! This method allows you to determine the Kd of WT and mutant proteins binding to DNA. The most convenient way to run the assay is to have the DNA labeled with a fluorophor (you could just order a DNA oligo with a Cy5 at the 5' end for example) and then you titrate in the protein. This way you would have one single oligo and you could test all your mutants!

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

If I can provide you information on the technology and how to run the experiment, please do not hesitat to contact me directly ([email protected])

there are several methods:

1. If instrument is available one could use isothermal calorimetry which gives quality data.

2. For fast screening type experiments, you can do ELISA assays. Purchase biotinylated oligo and immobilize them in 96-well plate coated with streptavidin. add your protein, incubate and wash. Detect the binding using antibody against your protein conjugated to HRP or any other enzyme or fluor. You can check many conditions at one shot.

3. Fluorescence anisotropy assay. you need labeled oligo only.

4. Again you can immobilize your DNA to a column/agarose beads using same biotin-streptavidin chemistry and look for binding by addition of your protein. Protein can be eluted by adding free unlabeled DNA and quantified.

I agree with the points above, esp. anisotropy studies. Analytical ultracentrifugation- this is a bit more tedious, but it gives you a lot of other information as well.

If you have an antibody against your DNA binding protein, you could try DNA binding ELISA, which is also quantitative.

All the methods mentioned above are solid approaches, I haven't seen this mentioned yet but you can also try crosslinking using UV, especially if you have an antibody to your protein.