Hi all,
I was wondering if there was a minimum kDa value that a protein would need to have in order to cause a visible gel shift in an EMSA assay. My protein of interest is rather small (15-20 kDa), so I wondered if the protein alone could cause a shift that would be visually obvious or if a supershift with an antibody might be needed in order to detect any difference.
Thank you!
Is this gel shift the same as the mobility shift seen on SDS-PAGE when comparing the sample run reduced and non-reduced?
Yes, if your protein actually binds DNA you have to be able to observe a clear shift. Obviously, It also depends on the size of your probe, but I think that 15-20 kDa are enough to determine a retardation of DNA running. I hope I have been helpful!
Hi Caroline
I haven't done EMSA (but that won't stop me offering an opinion or two!).
If the probe is synthetic, maybe you can choose the sequence such that there are multiple (putative) binding sites for your protein (assuming it binds to a specific sequence rather than just DNA in general).
If your protein is a non-specific DNA binding protein (and you're testing variants for their ability to retain binding activity, for example), then you'll have multiple binding events and a huge shift (or not, for the mutants).
As Nicola said, the size of the DNA molecule will have a big influence on what extent the protein band is up-shifted.
You could play around with the gel % to maximise the shift.
Don't most (?) target-specific DNA-binding proteins dimerise/oligomerise upon DNA binding? If yours does too, then the shift will be greater than the 15-20kDa you're expecting.
Good luck
Mike
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