Strange results

[hurricanemego]

I am performing EMSA using recombinant protein which I expressed in E. coli. I am getting shifts of my probe as expected. The unexpected part is that when I add in cold mutant oligos (200ng) to show that the mutant oligos do not bind and thus do not outcompete the labeled probe for protein binding I actually get more distinct bands that are sharper and bolder than when I use no cold oligo in the reaction. In theory the shift bands should look about the same between these two types of binding reactions. Can anyone think of any reasons why this might be happening, or how I might be able to fix it? I use 250ng of poly dIdC as my nonspecific DNA because higher amounts of it previously caused the intensity of my bands to decrease. Because I use counts per minute to determine how much probe to use, I can only estimate that I use somewhere between .5-1ng of labeled probe. Any suggestions...questions if this post is confusing?

Thanks!

[admin]

Hey Hurricane,
the mutant oligos, do they have mutations in them or are they the same as the E.coli sequence?

Also you are use 500X the amount of polydidC, which is quite high however should only have a small effect on your binding.

Your protein may be recognizing the cold competitor, or may not be as specific as you thought. Either that, or you may be misrepresenting the cold competitor in several ways:

1) have you compensated for the additional volume? Increased volume decreases the concentration of binding protein and sequence, thus decreasing the frequency of binding

2) have you checked that the cold-competitor/dIdC does not have DNase, or other inhibitory substances such as salts etc that may be inhibiting your reaction?

3) also you may be affecting the pH adding the cold competitor etc

There are many possibilities. have you narrowed it down?
good luck

[hurricanemego]

So my mutant oligos are actually mouse sequences that have had the potential binding sites for my protein mutated to a sequence that should be unrecognizable (basically i scrambled up the 6 bp core binding sites). I have tried different polydIdC concentrations and it doesn't really make a big difference in terms of the way things look. My PI says the stronger binding with the cold mutant competitor is not a big deal, as it is not a super strong increase in binding. I know that volume isn't a problem because I always keep the volume constant, and all conditions are kept the same between all the reactions so I don't know how pH or salt conditions would be changing with the addition of the mutant oligos considering that all oligos have been HPLC purified the same way. Anyways, thank you for you suggestions. I'm gonna keep working on it.