Gel shift assay

[FM07]

Hi, I am using Roche 2nd generation Gel shift assay kit, couldn't find shift. Want to try out Zn++ and Mg++, could anyone give me a suggestion of which concentration to start with?

Thanks

[admin]

Hello FM07,
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.

Cheers

[FM07]

Thank you for the reply. Sorry for the late response. The fact is that I was dragged away from this because the mice are ready for analysis. Is coming back, will try out your suggestions.

I am using the Hepes buffers, pH 7.9. My problem is that I don't have any positive control. We know next to nothing other than their potential binding sites. If gel shift assay isn't working out, we may have to go with ChIP.