Hi Rajendra

Please indicate on the attached gel what is in which lane. I am unsure which region your are referring to.

Jesper

here with I'm attaching a labelled image, in which the red circle region in the line 3 was related to my question. (from lane 1 to lane 3 protein conc increasing fro 0 to 300 picomoles)

I'm assuming the intense band in the circle is the one point in DNA which is excessively getting cleaved but i'm not getting why there were no bands above and below of it?

Hi Rajendra

I am not sure how much experience you have with this, so I apologize in advance if my suggestions seem trivial.

One possible explanation is that your transcriptional regulator might be contaminated by something that disturbs the activity of DNAse I. Try incubating the enzyme with a control DNA which shouldn't bind the transcriptional regulator. If you see a difference in the digestion pattern, then the problem lies with the protein.

Another problem might be excess digestion. In order for your footprint to look good, it is essential that each strand be cleaved only once by the DNAse. Add unspecific DNA in excess (1-2 ug DNA) to all of your samples and then run a test digestion to find optimal conditions for the DNAse

Finally, this cold spring harbor protocol should give you some valuable hints

http://www.ncbi.nlm.nih.gov/pubmed/18265087

good luck

Jesper

Hi there,

DNaseI footprinting is not a trivial method to use for identification of the binding site.

But in this particular case, it may be possible that the hypersensitive sites associated with the protein presence indicate conformational changes within the DNA upon formation of stable binary complex. Usually, binding-specific hypersensitive bands appear on both sides of the binding site. Similar to increase in protection of the corresponding binding site, you should see increase in intensity of the region(s) adjusted to it. In order to verify that what you see is not a cleavage due to nuclease contamination you may prepare a DNA template with 10-20 bp shifted binding site. Do DNaseI footprint on that construct and check the pattern of cleavage/protection. If you find a hypersensitive region shifted accordingly, than it is specific to the binding.

As an example I send you my DNase footprint where you can easily see both, efficient protection from DNaseI within the site of binding and increase in hypersensitivity on both sides of the binding site. The protein is p53. Many other transcription factors with high affinity are known to induce conformational changes upon binding, hence hypersensitivity band.

Good luck

DNaseI assay with clear hypersensitivity regions

Thank you Dear Jesper Nielsen for your suggestions. I will try doing with a non specific target and will check whether DNase able to digest the same in presence of my transcription regulator.

thank you dear Oleg Laptenko for your reply, you mentioned the two hypersensitive sites appear above and below which were commonly seen but in my case there is a single site visible and there after no bands seems to be appear..