Hello,
we are running sds-page electrophoresis gel of two purified protein. Both proteins have the same theoretical size but in the gels they migrated differently. Both have different Pi, one is 6 and the other one is 7. Do you think that this could affect the migration?
Thanks.
In theory, no. The SDS creates a fixed charge-to-mass ratio and electrophoretic separation should be on the basis of size. Other things to consider in this situation are (i) possible glycosylation (ii) proteolytic cleavage. How close in size are the proteins based on their amino acid sequences? What is the difference in mobility when checked against a series of molecular weight markers?
Migration can be affected by many things. We are working with intrinsically disordered proteins, and they always migrate at a molecular weight different from the theoretical one
I have observed differences in migration of the protein in SDS-PAGE just after phosphorylation, so many post-translational modifications may influence that, I guess.
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