I have a query about the formation of protein-DNA complexes:
I have a particular target sequence which is bound by a protein at a 2:1 ratio. Upon increasing the protein:DNA ratio to 5:1 and 10:1 respectively, the band shift I normally get at the 2:1 ratio seems to migrate slower with the addition of more protein. I am using a synthetic oligonucleotide, with ~6bp flanking the consensus. Is this just an artefact, or is there actually an interaction taking place in the presence of excess protein?
It can be also protein-protein interaction. How big is the protein?
Robert, It was originally a competitor free EMSA, with just the 2:1 complex. I will try and use some poly dC's, but it was just a curious experiment - we observe heavy smearing for an R->A mutant of my protein on EMSAS, and we seem to be able to pull the DNA up with a 50 Protein : 1 DNA ratio. However, in such cases, we DON'T see the supershift, just a movement of the smearing. So I was wondering if this supershift was artefactual. What's more, with a mutated DNA site which does not appear to bind in the normal 2:1 complex, the supershift occurs when we increase it to 10 protein :1 DNA.
Hey Benedict,
I was wondering if you have figured out your problem. I, too, am seeing a supershift with mutant oligo once the antibody is added and am not sure what could be causing this. Please let me know!
Thanks
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