Dear all,
my protein of interest appears at 27 kDa on SDS-PAGE (Novagen Novex NuPage Gel System, denaturing, Marker: GE life sciences low molecular weight marker) - calculated molecular weight is 37 kDa.
A rather strong difference of 10 kDa.
I am working with potentially infectious material, so I cannot use mass spec or other methods for molecular mass determination...
The protein contains some rare codons, might endure truncation: I expressed in CodonPlus, no change in molecular weight observed on SDS-PAGE.
The protein also contains loads of cysteins, and might not be completely unfolded/reduced on the gel - I added DTT/mercaptoethanol in combination to compensate - no change in electrophoretic mobility observed.
I am a bit desperate here, what can I do to check what I have there?
Could it be that it is the correct protein, but just running "odd"?
Thanks for any help,
Peter
Peter,
Well a lot could be happening here. The protein could be proteolytically modified reducing the expected molecular mass by SDS-PAGE... and/or the protein may run aberrant under SDS-PAGE conditions.
Aberrant migration is not that rare. SDS-PAGE is all about "relative" molecular mass and not the true molecular weight of the protein. The SDS essentially interacts with the protein to denature it and give it a net negative charge. If the protein interacts very well with SDS it may give you a protein that runs faster in the electrical field, conversely if the protein does not interact well with SDS it may run more slowly in the electrical field. Also, proteins that are more acid or basic (more negative or positive in charge) have been shown to run abnormally in SDS-PAGE systems.
Thanks for the reply, James.
I did an analysis of the sequence, I got:
Total number of negatively charged residues (Asp + Glu): 20 %
Total number of positively charged residues (Arg + Lys): 5 %
In addition, I have 13 % valine in the sequence. Hydrophobic residues should bind SDS more effectively?
A surplus of negative charges and a potentially high ability to bind SDS... This might explain why it runs further than it should?
Another possibility: Proteolysis, unfortunately, can explain migration at a lower than expected MW. Proteolysis can occur in vivo, or following cell lysis. A distinct band will occur if there is an especially open region of the protein.
I am expressing in BL21(DE3) - they are supposed to be essentially free of major proteases?
It is a good idea always to use protease inhibitors to prevent proteolysis when lysing cells. Probably no cells are completely free of proteases. If you know anything of the protein's structure you could see if there is a structurally open region that might be especially vulnerable to proteolytic attack, at the region 10 kDa from either end of the protein.
Now we are getting into the physical chemistry of protein-SDS interactions that are not completely understood. A wide variety of models have been proposed for the structure of the denatured protein-SDS aggregate, and the answer may be a mixture of many models. It is difficult to predict how a protein is going to interact with the detergent just based on the primary amino acid sequence and the net charge of the polypeptide. There are numerous forces at play including hydrogen bonding, Van Der Waals forces, ionic interactions, hyrophobic interactions, etc... all within the folded protein (not to mention between proteins). But, the short of it is that more negative changes on the protein than expected may cause the specific protein to migrate faster than expected under SDS-PAGE conditions resulting in what appears to be a "lower" than expected molecular mass.
A specific example that I have for you is the flagellar protein (FlaB) from Borrelia burgdorferi. This protein has the following predicted molecular mass and properties:
Molecular Weight 35763.83 Daltons (35.7 kiloDatlons)
336 Amino Acids
26 Strongly Basic(+) Amino Acids (K,R)
30 Strongly Acidic(-) Amino Acids (D,E)
117 Hydrophobic Amino Acids (A,I,L,F,W,V)
122 Polar Amino Acids (N,C,Q,S,T,Y)
5.548 Isolectric Point
-3.555 Charge at PH 7.0
But it migrates under SDS-PAGE conditions as 42-43 kiloDaltons, a difference of about 6 to 7 kiloDaltons. But there are similarly sized proteins with a similar pI that do not migrate aberrantly. So, the answer is more complicated than just pI and net charge. The order and juxtaposition of the amino acids likely plays a role in the interaction with SDS as well.
OK, interesting. I am definitely going to check on proteolysis.
How aggressive are the proteases left in BL21(DE3)?
I am doing the gels directly after lysis, normally there should not be enough time for proteases to work on the protein, and not that uniformly... (I get a single, distinct band).
Speaking of pI, it is 5.3 for my protein, which would result in a drastic negative charge at 6.8 (the pH I use for my running buffers in SDS-PAGE).
Might still be a possibility to explain the downshift of 10 - 8 kDa?
True that the physical properties of your protein may influence its migration under SDS-PAGE conditions and is a reasonable explanation as to why it is migrating at such a different mass than expected. Sometimes proteins with an acidic pI will run faster than expected and sometimes they run more slowly.
If you are worried about the loads of cysteins in your protein causing problems, you could always try reducing and then alkylate your sample using iodoacetamide. This would essentially cap the sulfhydryl groups and not allow disulfide bonds to form or reform during the running of the gel.
I use SDS-PAGE for confirmation of protein expression and to get some idea. For surety, I would recommend MALDI-TOF.
Hi
May I suggest few things that may help
Try different protein marker or pure protein of known MWt.
Does the loading mix turn yellow (acidic) during sample preparation? this will make protein sample migrate anomalously. if yes then add NaOH until turns to blue.
Good Luck
I today cleaved the sequence with a protease to get an idea where the truncation happens - the protein is seemingly cleaved at a rather unstructured domain in vivo (possibly the lack of structure provokes cleavage by proteases...)
Seems that I need to get rid of that unstructured sequence. It is part of a linker between two domains, so will not be easy.
Thanks for all your suggestions!
OK, I nailed the problem, it really was due to proteolysis. There is an unstructured region in my protein, where the cleavage seems to occur... Thanks for all the helpful hints and suggestions! :)
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