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[DiAzenha]

Dear all,

I am currently trying to set up the EMSA technique to study the impact of a promoter mutation on transcription factor binding.

I have been following Aimee's protocol, however I am not getting any shift.

I don't know the transcription factors that bind to that portion of the promoter (although I have done the bioinformatic prediction), so I don't know their molecular weight. I am running the reactions overnight at 50V with constant temperature set at 15ºC in a 10% polyacrilamide gel. For optimization purposes I am revealing with silver nitrate staining. However, once I see the shift, the detection will be done with avidin-HRP, since my oligos are biotin labelled.

I can see that the mutated oligo is consumed in the reaction, compared to the wild type, but I can't detect the shift.

I was wondering if anyone could give me some hints about what I can do to improve my EMSA.


Thank you!