Hi everybody
Since the beginning of my PhD I worked on a small Intrinsically disordered protein (13 Kda) which appear higher then it theoretical mass, and this is mostly due to it's N-terminal part. It's not a membrane protein. Since I use denaturing gel (SDS) would it be possible that the amino acid composition of the protein slow it down and make it look taller than it is.
Thank you
Yes, the amino acid composition of a protein can have an influence on how well the protein interacts with SDS. Mass determination by SDS Gel electrophoresis depends on the assumption that the number of SDS molecules bound is linearly dependent on the length of the unfolded protein and you therefore get the same mass to charge ratio for all proteins. If your protein binds less SDS, it carries less charge and therefore shows a higher apparent molecular weight.
How big is the size difference ?
While I do agree with Annemarie about the non linear relationship between SDS binding and protein size, you can expect post translational modifications like glycosylation, glycanation, ubiquitnation to increase the size of your protein.
Hi Ferdinand,
Yes, the amino acid composition does matter. For example, proteins with many negatively charged residues tend to show slower migration on a SDS-PAGE than expected (theoretical molecular weight). There are other reasons too for this altered migration which you may find here: Article Abnormal SDS-PAGE migration of cytosolic proteins can identi...
Good luck!
Anoop
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