[Nescafe]

Hello,

In western blot you denature the protein using SDS and the separation is solely based on size and not charge as all of the proteins will bare a negative charge. In EMSA you can use denaturing or non-denaturing conditions for the analysis.

Lets say in this scenario we are interested in investigating the binding of protein A to a specific sequence of DNA. We are running the gel under non-denaturing conditions as to preserve the native structure of the protein so it does not disintegrate and lose its affinity for the DNA sequence. There is also another protein (lets call it protein B) in the same cell extract that binds this exact sequence of DNA and it has a molecular weight that is right around the same kDA as protein A (88 v.s. 90 kDa). Charge wise, protein A is slightly positive while protein B has more of a negative charge but not too much as to repel DNA. These two should still separate due to their difference in charge as one will move faster towards the positive end of the gel while the other runs more slowly?

I am a bit surprised to learn that EMSA is able to separate Protein-DNA/RNA not only by size but charge as well under non-denaturing conditions and want someone to let me know if I am understanding this correctly. Previously I had thought that in EMSA (whether denaturing or non-denaturing) the separation is solely based on charge.

Thanks!

Nescafe.

[Yggdrasil]

Separation occurs because of a change in the shape of the complex. For example, the electrophoretic mobility of nucleosomes (complexes of histone proteins and DNA) will change depending on the position of the nucleosomes along the DNA (e.g. whether the nucleosome is on the end of the DNA or in the center).

I'm not so sure whether the charge of the protein matters much since the DNA carries such a large negative charge that the protein would have to be really highly charged to alter the net charge of the complex.