I have been expressing a protein with a tag which is then removed with a protease and repurified. Both purification steps use a Ni-NTA column. I run the samples on SDS-PAGE (15%, self-poured). With the tag, my protein should be 38.8 kDa, but seems to run at 50.8 kDa. After the digest, the protein should be 25.4 kDa, but is running at 30.5 kDa. The cleaved SUMO tag is 13.4 kDa, but runs at 14.9 kDa. (Note: It has been reported that the tag runs on SDS-PAGE at around 18 kDa.) What properties of a protein could cause it to run at a different size than expected? I am working with an intrinsically disordered protein, so maybe that caused a problem? It is rich in glycine, tyrosine, and serine.
Well, no one answered this, but I did find an answer eventually. First off, my protein of interest is a disordered protein. That means that it lacks a lot of the hydrophobic residues that we normally see in folded proteins. While it doesn't seem like that is an issue, the truth is that our standards are based on folded proteins, and without the hydrophobic residues, the protein runs differently. Generally, the disordered proteins run 5-10 kDa higher than they are expected. Just another joy of dealing with weird disordered stuff.
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