Maximum Oligonucleotide Size and Gel Percentage

[Ekn2484]

Hi. I am just starting EMSAs for the first time. My promoter sequence is 2.5 kb, and I tried to perform EMSA with this sequence. I found that my probe alone without any nuclear extract did not even enter the gel when I used a 6% gel. Does anyone know the maximum size of an oligo that can be used with EMSA. I realize that such a long sequence would bring many nuclear proteins with it and would possibly make the band very fuzzy, but is it possible to use a longer probe with EMSA? If so, what percentage gel and what conditions are favorable for this? Thanks.

[admin]

Hello,
wow that is tough its quite big. Why are you using this 2.5kb promoter sequence? You will need a smaller gel percentage and even possibly agarose gel.

Can you isolate a smaller binding element from the 2.5kb promoter using Matinspector or similar promoter analysis tool.

The problem is if you use non-denaturing gels with a 2.5kb promoter fragments dozens or hundreds of proteins would be binding this 2.5kb fragment and its unlikely to enter the gel.

I would use a much smaller fragment (even 200bp), to analyze this. If you really need to analyze a large fragment use a different method such as a DNAse protection assay.