Why is it that many proteins do not migrate on SDS-PAGE gels at their predicted size. What is the best way of determining that the band is due to the protein in question and not non-specific? Is using a known control good enough. I have used a purchased positive control and found that the protein was detected at a higher mw than bands from my sample but no other bands were detected. Can someone explain why that is?
I use 4-20% gradient gels and proteins from mostly mouse and rat brain tissue and both human and glioma cell lines

Murphy,

The rate of protein migration depends upon the net charge, size and shape. Thus any of these parameters will make a protein migrate differently at typically a higher apparent molecular weight.

Another possibility is aberrant glycosylation, which can make a protein run faster or slower than "controls" that might be expressed in a different system e.g., yeast vs E. coli vs mammalian cells. I suspect your "positive control" was expressed in a non-mammalian system, check the stat sheet. I have had this problem as well. You can also do a "Site Search" in the left corner of any page on the board for deglycosylation enzymes like PNGaseF that might help settle the issue.

The best controls are specific antibodies. Im not sure whether you are running a Western blot or stained gel to detect your protein, but there are many posts in this forum with discussions on the proper controls for both. I suggest trying the "Site Search" to look through them.

Let us know how things work out.

Rb

Like previously stated, glycosylation and phosphorylation will retard (no jokes please) the migration. Most of times, those two things are the reason for a higher molecular weight. There are kits out there to remove the attached parts.

I was confused over your comments. Are you saying that your protein standard is running differently as well?? If so, you might want to check if you are using the lammeli formulation or a bis-tris formulation buffer.

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