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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I am having this same problem as I am trying to detect a membrane protein. It is a receptor that has a MW of 43k Da. Initially, I did a RIPA buffer extraction of my protein from tissue and all I could detect was a trimer around 128 kDa and and a dimer but no band around 43 kDa. I even had the manufacturor send me a new antibody and I still got similar results. So I then did a membrane prep where I used 0.3 M sucrose to lyse open the tissue (in both buffers I added protease inhibitors). I then did a high speed spin to pellet down the membranes. Long story short, I still cannot detect anything at 43 kDa despite the fact that I am picking up trimers and dimers. I have found nothing that suggests that this membrane protein dimerizes (it is a transmembrane protein). As a caveat to all of this the protein is predicted to react with rat based on sequence homology. Any suggestions?

I have the sample problem: expected membrane protein (receptor) of 45 kb appear at 130-140 kb. I tried to not to boil, but I have the same problem
help please

Based off the replies in this string, everyone seems to be having the same problem. Proteins migrating aberrantly on gels and Western blots. First off proteins NEVER migrate at the predicted size. In the above string there are several suggestions as to how to reduce protein dimers and trimers as well as remove glycosylation to visualize protein bands on gels and Western blots. You all also have the problem of dealing with a membrane protein, which means that it is totally out of its native environment and likely refolds into a new structure to hide the hydrophobic domains usually in the membrane.

My suspicion is the bands you are seeing are the actual protein itself and not some strange dimer/trimer.

Try all the basic things

1. boil with a reductant like DTT or TCEP.
2. treat with PNGASE F or check for N-linked glycosylation sites by blasting your protein. Remember almost every protein that makes it out of the cell is glycosylated since it comes through the ER/golgi pathways.
3. try a new gel method such as the NuPAGE gels from Invitrogen or Bis/Tris gels.
4. Always run a no-primary control for the western blot to insure you are not looking at artifacts on the blot.

Good luck and let us know what you find out

RB