Help needed for DNase I Footprinting

[Ferland Louis H.]

On 27 Mar 1996, Dominic Voon wrote:

> Date: 27 Mar 1996 01:24:25 GMT
> From: Dominic Voon <dvoon at cyllene.uwa.edu.au>
> To: methods at net.bio.net
> Subject: Help needed for DNase I Footprinting
> 
> Hi all,
> 	I have been performing DNase I footprinting on a promoter 
> fragment using Jurkat (T-ALL) nuclear extracts for a while now. Using 
> crude extracts, I have been able to get significant protection though 
> incomplete. I have since been trying to get complete protection by 
> increasing the amount nuclear extract used and using less radiolabelled 
> probe (promoter fragment). So far, I have been puzzled by the fact that 
> although i have used up to 1/6 th the original amount of probes, I have 
> not been able to get six times the protection. I am beginning to think 
> that something is not right with my techniques, or there is some way I 
> can improve the quality of my nuclear extract. So, if any of you kind 
> souls out there has a good protocol for making crude nuclear extracts 
> from Jurkat or lymphoid cells, please email them to me. Also, if anyone 
> could shed some light into the problem that i am encountering, please 
> (please ! :-) let me know.
> 	Your help will be most appreciated.
> 
> warmest regards
> dom

Dom,

I did quite a lot of footprints, though this was many years ago. In my 
system, the protection was very good and I generated very clear 
footprints. However, I don't claim to have superior technique for those 
nice images. Rather,my assessment of the situation is that I have no 
reason to think that all DNA-binding proteins interact with their cognate 
site in such a manner as to fully protect it from DNase attack. Very 
often, one even sees bands whose intensity *increase* rather than be 
eliminated by the interaction. Your idea of increasing the extract/probe 
ratio is good, but I would consider the possibility that a complete 
washoff of all bands throughout the site may simply not be possible due 
the exact nature of the interaction. On the other hand, purification of 
the protein (even partial) sometimes brings up striking improvement. 
Still, you may not need completely blank footprints for publication of 
your results, though these are obviously more convincing!

Keep the morale!


Dr. Louis H. Ferland
Centre de Recherche, Hotel-Dieu de Montreal
Dept de Nutrition, Universite de Montreal
Phone: (514) 843-2757     FAX: (514) 843-2719