I am in the planning stages of conducting an EMSA and had a question on the DNA probe design. I am planning to test whether methylating a specific region will inhibit my protein of interest to bind there or not. Hence, my interest in an EMSA. The problem is that I'm interested in looking at a 400bp region that contains multiple CpG sites, where I can either methylate all the sites or preferentially methylate a select few.
Anyway, after researching various protocols, I have read that you should design your probe no longer than 200bp. My question is, does anyone have any experience doing an EMSA at a 400bp region and what would be the downsides of doing an EMSA on such a large region (if it's possible). I have read that you can fall into the trap of having too many proteins binding to such a large region, which is why you would avoid doing one. Are there any other reasons why this would not be such a good idea? To generate the 400bp probe, I am thinking to create 5' biotin labeled primers to amplify my region of interest.
Is there any other way to test this experiment other than dividing the 400bp region into seven 60bp regions and testing each separately? I want to avoid doing that because I'm interested in the region as a whole.
I did sevral EMSAs with RNA frgments from 200 - 700 nt. For such fragments I performed EMSAs in 5-6% PAA with 75:1 acrylamide/bisacrylamide ratio and everything worked fine (I obtained sharp bands and nice shifts). Alternatively you can use Filter Binding Assay where ribonucleoprotein complexes are retained on nitrocellulose membrane.
Thanks for your experiences, Leszek! It's good to hear that someone else performed an EMSA with a larger probe, although you did it with RNA.
Any other tips or experiences would be welcome!
In your case using DNA is in my opinion an advantage because it should migrate as a single conformer unlike RNA where quite often multiple structural conformers are visible during native conditions.
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