I have Problem with Gel ratardations. I am using DIG-Labelled DNA and a putative Bindingprotein to achieve a Gel Shift. The Protein should only bind if it is Phosporylated.
So this is how i made the experiment.
A 6% TBE Gel with the following application DNA without Protein DNA and unphosphorylated Protein and finally DNA+ phosphorylated Protein.
And this is what i get
The signal without Protein and +phosphorylated Protein run at the same height so no interaction can be proven but the siganl of +unphosphorylated Protein runs much further.
That doesnt make sense to me because the DNA should not run further than the control (without Protein) since the size of the DNA does not vary.
I am not sure if the concept of Gel Shift is clear. A certain DNA runs a certain length in the gel. Now if the binding protein binds to the DNA the DNA runs shorter and thats the shift.
Anyhow, from my understanding of the theory of the experiment, rather thand hands on experience, my 2 cents are:
DNA migration in Agarose is not directly proportional to the length of the DNA fragment, but by its bulk. So DNA that would be tightly packed would migrate farther than a DNA of the same length completely unpacked. The electric charge (load?) of the molecule is also important
Hence if your protein tighten DNA around itself in its unphosphorylated form, or if your protein is negatively charged in your migration conditions you may end up with a complex that is faster than the DNA itself.