| | Re: Gel shift assay
welcome to the forums.
I have been doing shifts for years and its one of the most tricky techniques in molecular biology.
Do you have your binding protein purified? I was not so lucky, if so make sure you use it as a positive control.
If not, there are several steps of optimizing. Would be a good idea to have a large gel or two and run lanes of different binding buffers you prepared, even increasing Mg++ and Zn++ concentrations.
From my experiences, Mg++ and Zn++ is important for binding however the most important things you should consider first you are not getting binding are salt concentration, buffer type and its pH.
You should try varying these before optimizing Magnesium and Zinc. What I would do is use a buffer close to what is working for others and including Mg and Zn. Then you can fine tune your buffers for your protein of interest.
Usually people are using Hepes buffers, pH around 7.7-8.2 pH's and throwing in Dnase inhibitors or Rnase inhibitors, and then adding some Mg and Zn. Check out some papers on pubmed for exact concentrations. I would look for your protein or the analysis of a similar protein (or another protein in your protein family of interest) for clues.